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| United States Patent |
5,252,474
|
|
Gewain
, et al.
|
October 12, 1993
|
Cloning genes from Streptomyces avermitilis for avermectin biosynthesis
and the methods for their use
Abstract
There are disclosed plasmids containing DNA isolated from Streptomyces
avermitilis, the microorganism which is used to prepare avermectin
compounds, identified as pAT1, pVE650 pVE855, pVE859, pVE1446, pVE923, and
pVE924 which contain the genetic information for the biosynthesis of the
avermectins. Methods for the isolation of such plasmid and for the
manipulation of the plasmids to alter the formation of the avermectin
compound are also disclosed.
| Inventors:
|
Gewain; Keith M. (Middelsex, NJ);
MacNeil; Douglas J. (Westfield, NJ);
MacNeil; Tanya (Westfield, NJ);
Paress; Philip S. (Maplewood, NJ);
Ruby; Carolyn L. (Montclair, NJ);
Streicher; Stanley L. (Verona, NJ)
|
| Assignee:
|
Merck & Co., Inc. (Rahway, NJ)
|
| Appl. No.:
|
490723 |
| Filed:
|
March 14, 1990 |
| Current U.S. Class: |
435/91.1; 435/76; 435/119; 435/252.33; 435/252.35; 435/320.1; 435/486; 435/488; 435/489 |
| Intern'l Class: |
C12N 015/00; C12N 015/11; C12P 017/18 |
| Field of Search: |
435/71.2,71.3,320.1,172.3,252.3,76,252.33,119,252.35,886
935/9,29,72,73,75
536/27
|
References Cited
U.S. Patent Documents
| 4703009 | Oct., 1987 | MacNeil et al. | 435/172.
|
| Foreign Patent Documents |
| 118367 | Sep., 1984 | EP.
| |
| 173327 | Mar., 1986 | EP.
| |
| 204549 | Dec., 1986 | EP.
| |
| 276103 | Jul., 1988 | EP.
| |
| 276131 | Jul., 1988 | EP.
| |
| 62-195286 | Feb., 1986 | JP.
| |
| 62-224292 | Mar., 1986 | JP.
| |
| 8703907 | Jul., 1987 | WO.
| |
Other References
The Extended Phenotype, 1982, Dawkins, Oxford University Press, Oxford, pp.
85, 86, and 287.
Hutchinson; Applied Biochemistry and Biotechnology 16: 169 (1987).
Murooka et al; Agric. Biol. Chem. 47: 1807 (1983).
Hopwood, et al Nature 314, pp. 642-644.
Schulman, et al, Jour. of Antib. 38 pp. 1494-1498 (1985).
Ruby, et al Sixth Annual Symposium on Actinomycetes Bio. pp. 279-280
(1985).
Ikeda, et al J. Bacteriol. 169, pp. 5615-5621 (1987).
Chater, et al EMBO Jour. 4, pp. 1893-1897 (1985).
Malpartida, et al Nature 309 pp. 462-464 (1984).
Schulman, et al Antimicrobial Agents and Chemotherapy 31 pp. 744 and 746
only (1987).
Fishman, et al Proc. Natl. Acad. Sci. USA 84 pp. 8248-8252 (1987).
Motamedi, et al Proc. Natl. Acad. Sci. USA 84 pp. 4445-4449 (1987).
McNeil, J. Microbiol. Methods 5 pp. 115-123 (1986).
Feitelson, et al J. Gen. Microbiol. 131 pp. 2431-2441 (1985).
Murakami, et al Mol. Gen. Genet. 205 pp. 42-50 (1986).
MacNeil, et al J. Industrial Microbiol. 2 pp. 209-218 (1987).
Chen, et al Bio/Technology 6 pp. 1222-1224 (1988).
Miller, et al Antimicrobial Agents and Chemotherapy 15 pp. 368-371 (1979).
Ruby et al, "Isolation and Characterization of Streptomyces Aremitilis
Mutants Defective in the Methylation of the Avermectins"--Conference Paper
(1980)--Abstract only.
|
Primary Examiner: Martinell; James
Attorney, Agent or Firm: Rose; David L., DiPrima; Joseph F.
Parent Case Text
CROSS REFERENCE TO RELATED APPLICATIONS
This case is a continuation-in-part of our application Ser. No. 390,576
filed Aug. 7, 1989, now abandoned, which in turn is a continuation-in-part
of application Ser. No. 331,146, filed Mar. 31, 1989 now abandoned.
Claims
What is claimed is:
1. Plasmid pAT1 (44.05 kb), pVE650, pVE923, pVE924, pVE855 and pVE859, and
pVE1446.
2. The plasmid of claim 1 which is pAT1 (44.05 kb).
3. The plasmid of claim 1 which is pVE650.
4. The plasmid of claim 1 which is pVE923, pVE924, pVE855, or pVE859.
5. The plasmid pVE1446.
6. The DNA responsible for avermectin biosynthesis contained on plasmid
pAT1 (44.05 kb), pVE650, pVE923, pVE924, pVE885, pVE859, or pVE1446.
7. A method for improving the yields of avermectin compounds from
fermentation broths containing a microorganism capable of producing
avermectin compounds which comprises incorporating one or more of the
plasmids pAT1, (44.05 kb) pVE650, pVE923, pVE924, pVE855, pVE859, or
pVE1446 or BamHi restriction fragments from said plasmids into such
microorganism.
8. The method of claim 7 wherein the microorganism is Streptomyces.
9. The process of claim 8 wherein the microorganism is Streptomyces
avermitilis.
10. The method of claim 8 wherein the microorganism is Streptomyces
hygroscopicus.
11. The process of claim 8 wherein the microorganism is Streptomyces
cyanogriseus.
12. The process of claim 8 wherein the microorganism is Streptomyces
thermoarchaenosis.
13. The method of claim 7 wherein the plasmid is pAT1 (44.05 kb).
14. The method of claim 7 wherein the plasmid is pVE650.
15. The method of claim 7 where the plasmid is pVE923, pVE924, pVE855 or
pVE859.
16. The method of claim 7 where the plasmid is pVE1446.
17. A process for the isolation of DNA from microorganisms comprising:
a) constructing a cosmid library of DNA from a microorganism in Escherichia
coli;
b) preparing filters which contain DNA from said cosmid library;
c) incorporating .sup.32 P into a purified Bam HI restriction DNA fragment
comprising a portion of the DNA responsible for avermectin biosynthesis
wherein said Bam HI fragment is contained on a plasmid selected from the
group consisting of pAT1 (44.05 kb), pVE650, pVE923, pVE924, pVE855,
pVE859, and pVE1446;
d) using the .sup.32 P containing DNA of step c) as a probe in DNA--DNA
hybridization with the filter from step b); and
e) isolating the cosmid DNA from a replica of the colony which hybridized
to the .sup.32 P containing DNA of step c).
18. A process of claim 17 wherein the microorganism is Streptomyces.
19. The process of claim 17 wherein DNA fragments are prepared from
plasmids pAT1 (44.05 kb), pVE650, pVE923, pVE924, pVE855 or pVE859 or
pVE1446.
20. The process of claim 17 wherein the Bam Hi restriction fragments are
prepared from plasmid pAT1.
21. The process of claim 17 wherein the Bam Hi restriction fragments are
prepared from plasmid pVE650.
22. The process of claim 17 wherein the Bam Hi restriction fragments are
prepared from plasmids pVE923, pVE924, pVE855 or pVE859.
23. The process of claim 17 wherein the Bam Hi restriction fragments are
prepared from plasmid pVE1446.
24. A process for the isolation of avermectin genes from Streptomyces
avermitilis which comprises the complementation of Streptomyces
avermitilis mutants with cloned Streptomyces avermitilis DNA wherein the
cloned Streptomyces avermitilis DNA is contained on a plasmid selected
from the group consisting of pVE650, pAT1 (44.05 kb), pVE923, pVE924,
pVE855, pVE859, and pVE1446.
25. The process of claim 24 wherein the plasmid is pAT1 (44.05 kb).
26. The process of claim 24 wherein the plasmid is pVE650.
27. The process of claim 24 wherein the DNA is contained on plasmid pVE923,
pVE924, pVE855 or pVE859.
28. The process of claim 24 wherein the DNA is contained on plasmid
pVE1446.
29. The microbiological strain Streptomyces lividans containing plasmid
pAT1 (44.05 kb).
30. A microbiological strain of claim 29 which is MA6619 (ATCC 67820) and
mutants thereof.
31. The microbiological strain Streptomyces lividans containing plasmid
pVE650.
32. A microbiological strain of claim 31 which is MA6618 (ATCC 67819) and
mutants thereof.
33. A microbiological strain Escherichia coli containing plasmid pVE923.
34. A microbiological strain of claim 33 which is MB5373 (ATCC 67891) and
mutants thereof.
35. The microbiological strain Escherichia coli containing plasmid pVE924.
36. The microbiological strain of claim 35 which is MB5374 (ATCC 67892) and
mutants thereof.
37. The microbiological strain Escherichia coli containing plasmid pVE855.
38. The microbiological strain of claim 37 which is MB5370 (ATCC 67889) and
mutants thereof.
39. A microbiological strain Escherichia coli containing the plasmid
pVE859.
40. The microbiological strain of claim 39 which is MB5372 (ATCC 67890) and
mutants thereof.
41. A microbiological strain Escherichia coli containing the plasmid
pVE1446.
42. The microbiological strain of claim 41 which is MB5472 (ATCC 68250) and
mutants thereof.
43. A Bam HI restriction fragment comprising a portion of the DNA
responsible for avermectin biosynthesis wherein said Bam HI fragment is
contained on pAT1 (44.05 kb).
44. A Bam HI restriction fragment comprising a portion of the DNA
responsible for avermectin biosynthesis wherein said Bam HI fragment is
contained on plasmid pVE650.
45. A Bam HI restriction fragment comprising a portion of the DNA
responsible for avermectin biosynthesis wherein said Bam HI fragment is
contained on a plasmid selected from the group consisting of pVE923,
pVE924, pVE855, and pVE859.
46. A Bam HI restriction fragment comprising a portion of the DNA
responsible for avermectin biosynthesis wherein said Bam HI fragment is
contained on plasmid pVE1446.
Description
BACKGROUND OF THE INVENTION
Streptomyces are producers of a wide variety of secondary metabolites,
including most of the commercial antibiotics. Because of this,
considerable effort has been invested in developing gene cloning
techniques for Streptomyces. Procedures for the efficient introduction of
DNA into Streptomyces by polyethylene glycol (PEG) mediated transformation
have been developed. Vectors have been constructed which include phages,
high copy number plasmids, low copy number plasmids and E.
coli-Streptomyces shuttle vectors. Numerous drug resistance genes have
been cloned from Streptomyces species and several of these resistance
genes have been incorporated into vectors as selectable markers. A review
of current vectors for use in Streptomyces is Hutchinson, Applied
Biochemistry and Biotechnology 16 pg 169-190 (1988). In many cases, genes
for the production of secondary metabolites and genes encoding for
resistance have been found to be clustered. Thus one strategy for cloning
genes in a pathway has been to isolate a drug-resistance gene and then
test the adjacent DNA for other genes for that particular antibiotic.
Examples of biosynthetic genes clustered near a drug resistance gene
include actinorhodin (Malpartida and Hopwood, Nature 309 pg 462 (1984)),
tetracenomycin C (Motamedi and Hutchinson, Proc. Natl. Acad. Sci. USA 84
pg 4445-4449 (1987)), and bialaphos (Murakami et al, Mol. Gen. Genet. 205
p 42-50 (1986 ), EP 173,327). EP 204,549 exploits the clustering of
drug-resistance genes and biosynthetic genes and claims a method for
isolating antibiotic genes by using a easily isolated drug-resistance
gene. Patent publication wo87/03907 discloses a method for isolating
polyketide antibiotics using cloned genes for polyketide synthase. This
application also discloses the cloning of genes involved in milbemycin
biosynthesis, a compound structurally related to the avermectins. Another
strategy for cloning genes for the biosynthesis of commercially important
compounds has been complementation of mutants. A library of DNA from a
producing organism is introduced into a nonproducing mutant and the
transformants are screened for the production of the compound. This
approach has also identified gene clusters involved in antibiotic
production, in some cases all the genes for the production of several
antibiotics have been cloned. In addition to the three examples above,
other examples of cloned Streptomyces genes involved in antibiotic
biosynthesis include tylosin (Fishman et. al., Proc. Natl. Acad. Sci. USA,
84 pg 8248-8252 (1987), undecylprodigiosin (Feitelson, et al., J. Gen.
Micro. 131 pg 2431-2441 (1985), methylenomycin (Chater and Bruton, EMBO J
4 pg 1893-1897 (1985), nosiheptide (JP 8636216) and Cephamycin C (Chen et
al., Bio/Technology 6 pg 1222-1224 (1988), JP 8667043). In several cases
new analogs of antibiotics have been produced by the introduction of
cloned genes into other Streptomyces (Floss, Biotechnology 5 pg 111-115
(1987), Hopwood et al., Nature 314 pg 642-644 (1985)). In other cases the
introduction of extra copies of biosynthetic genes into the original
producing organism has resulted in increased titer of the antibiotic. EP
238323 discloses the process of introducing a gene for the rate limiting
enzyme into the producing organism to increase titer of the antibiotic.
Streptomyces avermitilis produces avermectins, a series of 8 related
compounds with potent anthelmintic and insecticidal activity (U.S. Pat.
Nos. 4,310,519 and 4,429,042). A semisynthetic derivative of avermectin,
ivermectin, is a commercially important anthelmintic. U.S. Pat. No.
4,310,519 describes a mutant of S. avermitilis which lacks the furan ring
of the natural avermectins. Schulman et al., J. antibiot. 38 pg 1494-1498
(1985) describes a mutant, Agly-1, which produces avermectin aglycones A1a
and A2a. Ruby et al., Proceedings of the 6.sup.th International Symposium
on the Biology of Actinomycetes, G. Szabo, S. Biro, M. Goodfellow (eds.),
p.279-280 (1985) and Schulman et al., Antimicr. Agents and Chemother. 31
pg 744-747 (1987) describe 2 classes of S. avermitilis mutants, one class
is defective in O-methylation at C-5 and the other class is defective in
O-methylation at C-3" and C-3'. EP 276103 describes a mutant of S.
avermitilis defective in branch chain fatty acid dehydrogenase. EP 276131
describes a S. avermitilis mutant defective in C-5, C-3", and C-3'
O-methylation. Ikeda et al., J. Bacteriol. 169 pg 5615-5621 (1987), have
described the isolation and genetic analysis of two classes of S.
avermitilis mutants. AveA mutants were defective in avermectin aglycone
formation and AveB mutants failed to synthesize or attach the oleandrose
moiety to avermectin aglycone. They obtained genetic evidence that the two
classes of mutations are linked. This application describes the cloning of
genes required for the biosynthesis of avermectins. Other microorganisms
that produce avermectin-like-compounds are S. hygroscopicus, S.
cyanogrieseus and S. thermoarchaenosis. Such microorganisms may be
subjected to the same procedures as are described herein for S.
avermitilis.
SUMMARY OF THE INVENTION
Mutants of S. avermitilis which produce analogs of the 8 major avermectins,
mutants which produce avermectins in a different ratio than the original
soil isolate, and mutants which fail to produce avermectins, were isolated
from mutagenized cells. A gene library of S. avermitilis DNA was made in
the low copy number Streptomyces vector pIJ922. After ligation, the
resulting molecules were transformed into S. lividans and transformants
were selected as thiostrepton resistant (Thio.sup.r). Transformants were
pooled and plasmid DNA was isolated. Aliquots of the pIJ922 library were
introduced into mutants of S. avermitilis. Several plasmids were
discovered which complemented the defect in an avermectin C-5
O-methyltransferase (OMT) deficient strain. The first plasmid, pAT1, was
characterized extensively. Another plasmid, pVE650, was discovered which
complemented the defect in an avermectin aglycone producing mutant, this
mutant is defective in synthesis or addition of oleandrose moiety to the
avermectin aglycone. Subcloning analysis of pAT1 revealed the gene for OMT
was located on a 3.4 kb BamHI fragment. Subcloning analysis of pVE650
revealed that two Bg1 II fragments complemented the defect in several
avermectin aglycone producing strains.
Southern hybridization analysis of pAT1 and pVE650 indicated that the two
plasmids do not contain any overlapping sequences. However, it was
surprisingly discovered that the two plasmids contain regions of
non-exact, but related homology. Two different groups of related sequences
were discovered. The number of different BamHI fragments from the genome
of S. avermitilis that are in each group was determined by a Southern
analysis of the BamHI digested S. avermitilis genomic DNA. Seven separate
BamHI fragments showed homology to a probe from Group 1. These 7 were
composed of 1 fragment located on the DNA cloned on pAT1, one fragment on
the DNA cloned on pVE650, and 5 other bands located elsewhere in the
chromosome. At least 14 BamHI fragments contained regions of homology to a
probe from Group 2. Five fragments of pVE650, 1 fragment of pAT1, and 8
fragments elsewhere in the genome of S. avermitilis contained regions
homologous to a Group 2 probe.
A second gene library of S. avermitilis DNA was made in the E. coli cosmid
vector pVE328. Restriction fragments from pVE650 were used as probes to
isolate clones from the pVE328 cosmid library that contained sequences
homologous to pVE650. A series of cosmid clones were isolated that
collectively span over 110 kb of genomic DNA. This DNA includes the
avermectin C-5 O-methylransferase gene, the C-22, C-23 dehydrase gene,
several genes involved in avermectin aglycone formation, and at least 7
genes involved in the synthesis or attachment of oleandrose to avermectin
aglycone.
One region of the avermectin gene cluster was missing from the cosmids that
were isolated. This region was cloned directly from the S. avermitilis
genome into E. coli using an integration vector. An additional 15 kb of
the avermectin gene cluster was cloned this way.
DETAILED DESCRIPTION OF THE INVENTION AND ITS PREFERRED EMBODIMENTS
ISOLATION AND CHARACTERIZATION OF AVERMECTIN MUTANTS
Mutants defective in avermectin biosynthesis were detected by a thin layer
chromatography (TLC) screen of methanol extracts derived from
fermentations of single colony survivors of mutagenic treatments. Spores
of the parental strains were mutagenized with UV or NTG as described in
Hopwood et al., Genetic manipulations of Streptomyces-A Laboratory manual,
John Innes Institute. Norwich. 1985. The survivors of the treatment, which
killed 99% to 99.9% of the spores, were allowed to form well sporulated
colonies on Medium A. Spores from a single colony were inoculated into
0.25 ml of growth medium. After 40 hours of growth, 0.8 ml of fermentation
medium was added and the fermentation continued for 13 days. Fermentations
were incubated at 28.degree. C. on a rotary shaker at 220 rpm. Various
growth media and fermentation media can be used, several media have been
described in U.S. Pat. Nos. 4,310,519 and 4,378,353. A growth medium which
is particularly useful is Medium B, and a fermentation medium which is
particularly useful is Medium C.
______________________________________
Medium A
KNO.sub.3 1 g
Yeast Extract (Difco) 1 g
Malt Extract 1 g
Sodium Citrate 0.5 g
MgSO.sub.4.7H.sub.2 O 0.25 g
Trace Elements 2.5 ml
Glucose 2 g
Distilled water 1000 ml
Adjust to pH 7.0 with NaOH
Trace elements for Medium A contain per liter of
distilled water: 49.7 ml HCl (37.3%), 61.1 g
MgSO.sub.4 7H.sub.2 O, 2.0 g CaCO.sub.3, 5.4 g
FeCl.sub.3.6H.sub.2 O, 1.44 g ZnSO.sub.4.7H.sub.2 O, 1.11 g
MnSO.sub.4 H.sub.2 O, 0.25 g CuSO.sub.4 5H.sub.2 O, 0.062 g
H.sub.3 BO.sub.3, and 0.49 g Na.sub.2 MoO.sub.4.H.sub.2 O.
Medium B
MgSO.sub.4.7H.sub.2 O (12.5% solution)
2.67 ml
NaCl (12.5% solution) 2.67 ml
MnSO.sub.4.H.sub.2 O (0.5% solution)
0.67 ml
ZnSO.sub.4.7H.sub.2 O (1.0% solution)
0.67 ml
CaCl.sub.2.H.sub.2 O (2.0% solution)
0.67 ml
FeSO.sub.4.7H.sub.2 O (2.5% solution)
0.67 ml
KNO.sub.3 1.33 g
Hy-Case S.F. (Humpko) 13.3 g
Yeast Extract (DIFCO) 13.3 g
Glucose 19.95 g
Distilled Water 1000 ml
Adjust pH to 7.0 with 1N NaOH
Medium C
Peptonized Milk 20 g
Ardamine pH 4 g
Glucose 105 g
MgSO.sub.4.7H.sub.2 O 0.5 g
CuSO.sub.4.5H.sub.2 O 0.06 g
ZnSO.sub.4.6H.sub.2 O 1 mg
CoCl.sub.2.6H.sub.2 O 0.1 mg
FeCl.sub.2.6H.sub.2 O 3 mg
Add Distilled water 900 ml
to a volume of
Adjust to pH 7.2 w/1 N NaOH
______________________________________
Separately prepare a glucose solution of 35 g of glucose in a final volume
of 100 ml of distilled water, adjust pH to 7.2. After autoclaving add the
glucose solution to complete Medium C.
The fermentation broth was extracted with an equal volume of methanol and a
sample was applied to a TLC plate to separate the avermectins. The TLC
system employed separates the avermectins into 4 bands detected by UV
fluorescence quenching as described by Miller et al., Antimicrob. Agents
and Chemother. 15, 368-371, (1979). Extracts were spotted on E. Merck
Silica Gel 60 F-254 plates and developed in a solvent of Dichloromethane:
Ethyl Acetate: Methanol 9:9:1. In this system, the order of the
avermectins from fastest to slowest band is avermectin A1, A2, B1, and B2;
the a and b series are not resolved. Colonies showing compositional
changes, bands of altered mobility, or the absence of some or all
avermectin bands were repurified and refermented. Mutants which were
stable and gave reproducible fermentations were saved. The avermectins
produced by some of the mutants were isolated by preparative TLC and HPLC
and characterized by NMR or mass spectroscopy when necessary for
identification. In most cases identification was established through
direct comparison with pure samples of individual components (e.g. Bla for
identifying mutants deficient in O-methyltransferase activity) or modified
avermectins (e.g. avermectin aglycones or, desmethylavermectins) obtained
through chemical modification or fermentations in the presence of
inhibitors (Schulman et al., J. Antibiot. 38 1494-1498 (1985)). Table 1
presents a summary of the mutant classes isolated.
TABLE 1
__________________________________________________________________________
Avermectin Mutants of Streptomyces avermitilis
Mutant Class Fermentation Products
__________________________________________________________________________
Non-producers None
Aglycone producers (AGL).sup.1
Avermectin aglycones
Oleandrose synthesis
TDP-Oleandrose tranferase(s)
Avermectin C.sub.5 --O-methyl-
Avermectin B1(a + b) + B2(a + b)
transferase (OMT).sup.1
TDP-demethyloleandrose 3-O-methyl-
3', 3" Demethyl avermectins
transferase (GMT).sup.1
Dehydrase Avermectins A2(a + b) + B2(a + b)
Furan ring Defurano avermectins
__________________________________________________________________________
.sup.1 AGL: aglycone producer; OMT: Omethyltransferase; GMT: glycosyl
Omethyltransferase
The classified mutants were those where the blocks occurred after the
formation and closure of the macrolide ring structure, since detectable
fermentation products accumulated which could be isolated and identified.
These include two types of O-methyltransferase mutants. The first class is
defective in methylation at the C-5 position (avermectin
O-methyltransferase) and results in the accumulation of avermectin B
components (OMT.sup.- phenotype) Ruby et al., Proceedings of the 6.sup.th
International Symposium on the Biology of Actinomycetes, G. Szabo, S.
Biro, M. Goodfellow (eds.), p. 279-280 (1985) and (Schulman et al.,
Antimicr. agents and Chemother. 31 pg 744-747 (1987)). The second class is
deficient in methylation of the oleandrose moieties (glycosyl
O-methyltransferase) resulting in the accumulation of desmethylavermectins
(GMT.sup.- phenotype) Ruby et al., Proceedings of the 6.sup.th
International Symposium on the Biology of Actinomycetes, G. Szabo, S.
Biro, M. Goodfellow (eds.), p. 279-280 (1985) and (Schulman et al.,
Antimicr. agents and Chemother. 31 pg 744-747 (1987)). Biochemical studies
have indicated that these methylation reactions are catalyzed by at least
two distinct enzymes. A large class of avermectin mutants are unable to
synthesize or attach the oleandrose moiety to avermectin aglycones. These
mutants accumulate the avermectin aglycones and are defective in either
the synthesis of oleandrose diphosphonucleotide or the transfer of its
oleandrose moiety. Other characterized mutants include those unable to
close the furan ring and those with reduced ability to catalyze the
conversion of avermectin "2" precursors to avermectin "1" precursors. The
latter mutants accumulate primarily the avermectin "2" components and are
presumed to lack "avermectin" dehydrase activity.
The class of avermectin non-producing mutants presumably represents many
different blocks in the early steps of avermectin synthesis. These mutants
may be producing enzyme bound intermediates but do not appear to
accumulate any diffusable intermediates or U.V. absorbing material capable
of being transformed into avermectins. Pair-wise fermentations of these
non-producing mutants did not lead to the production of avermectins.
Methanol extracts of non-producers did not contain precursors able to be
converted into avermectins. Thus the avermectin non-producers have not yet
been classified into different groups.
Mutants unable to methylate avermectin at the C-5-hydroxy position produce
avermectins B1 (a+b) and B2 (a+b) almost exclusively. These mutants have
low or undetectable levels of avermectin OMT, an enzyme which utilizes
S-adenosylmethionine as the methyl donor (Schulman et al., Antibiot. 38
1494-1498 (1985). The levels of A components found in some mutants are
related to the leakiness of the defect since low but detectable amounts of
enzyme were also found to be present. The mutant phenotype appears to be
closely correlated to the lack of the OMT enzyme. Since the overall
avermectin titer of these mutants is unchanged from that of the parental
strain, it is likely that mutations responsible for the OMT phenotype are
structural gene lesions. Among the avermectin mutants isolated, the OMT
class is the best characterized and understood. This class was used first
in the complementation screen.
CLONING THE GENE FOR C-5 AVERMECTIN O-METHYLTRANSFEREASE
A genomic library in the low copy-number Streptomyces vector pIJ922
(Hopwood et al., Genetic Manipulations of Streptomyces a Laboratory
Manual, John Innes Institute, Norwich, 1985) was used for the mutant
complementation screen. The library was constructed by ligating S.
avermitilis DNA, which had been partially digested with Sau3A and size
fractionated, into pIJ922, which had been linearized with BamHI
restriction enzyme and treated with calf intestinal alkaline phosphatase.
The ligated DNAs were then transformed into either S.lividans or S.
avermitilis and thiostrepton resistant colonies were selected. Sporulated
colonies were harvested in bulk, diluted into YEME medium (Hopwood et al.,
Genetic Manipulations of Streptomyces a Laboratory Manual, John Innes
Institute, Norwich, 1985) and cultured for plasmid purification. The
purified plasmid preparations from these cultures constitute the pIJ922-S.
avermitilis genomic library. A representative number of initially
transformed colonies and those derived from a transformation using the
purified library plasmid preparations were checked for insert frequency
and size. The frequency of plasmids containing inserts was greater than
65% with an average size of about 20 kb. Neither the frequency nor that
average insert size differed significantly between the initial set of
transformants and that obtained with library DNA.
The library was initially screened by transforming avermectin
O-methyltransferase deficient (OMT.sup.-) mutant, MA6233, with library DNA
selecting for Thio.sup.r transformants. Individual transformed colonies
were scored for avermectin production and composition. The OMT mutant
produces only two avermectin TLC bands under these conditions (B1 and B2).
One OMT positive transformant was detected from screening over 10,000
transformants. This transformant was purified, retested for
complementation and the plasmid from one of the repurified colonies was
designated pAT1. Plasmid pAT1 complemented the OMT phenotype of all 6 OMT
mutants tested. A restriction map of pAT1, which contains 20 kb of S.
avermitilis DNA, was determined. The location of sites is presented in
Table 3 and the map is indicated in FIG. 1. The gene for
O-methyltransferase was designated avrA. Plasmid pAT1 was transformed into
S. lividans, and the resulting strain designated MA6619 which has been
deposited as ATCC 67820 at the American Type Culture. Collection, 12301
Parklawn Dr., Rockville, Md. 20852.
The OMT gene was localized by subcloning pAT1 BamHI fragments into pIJ922,
followed by transformation and complementation analysis. A subclone pAT83
containing the 3.4 kb BamHI fragment, was able to complement MA6233
(OMT.sup.-), indicating that the gene maps within this fragment.
CLONING GENES OF OLEANDROSE SYNTHESIS AND/OR TRANSFER
Subsequent to the isolation of the pAT1 an additional screening effort was
undertaken to isolate plasmids that would complement other avermectin
mutants. Aliquots of the pIJ922 library were transformed into MA6278
(AGL.sup.-, OMT.sup.-). Transformants were screened for the production of
glycosylated avermectins. This effort led to the isolation of pVE650, a
plasmid containing an insert of about 24 kb that complements a number of
mutant strains defective either in the synthesis of oleandrose or its
transfer to avermectin aglycone. Table 2 shows that pVE650 complemented
the defect in 21 aglycone producing mutants but five aglycone producing
mutants were not complemented indicating that some glycosylation genes are
outside of this cloned region. Neither pVE650 nor pAT1 complemented an
avermectin non-producing mutant or a GMT.sup.- mutant. A restriction map
was established for the pVE650 insert (FIG. 2) and Table 4 presents the
location of the restriction sites. A comparison with the map of pAT1 does
not indicate any fragment in common between the two clones. This plasmid
was introduced into S. lividans and the resulting strain designated
MA6618. This strain has been deposited as ATCC 67819.
Complementation analysis of pVE650.
Three avermectin genes involved in oleandrose synthesis or attachment have
been identified by complementation studies with pVE650, and two subclones.
pVE650 is a plasmid with a 24 kb insert of S. avermitilis DNA which
contains genes for the synthesis and/or addition of oleandrose to the
avermectin aglycone. A subclone, pVE806, which is pIJ922 with the 4.28 kb
Bg1II fragment cloned into the BamHI site, was found to complement some
aglycone producing mutants (Class I in Table 2). Another subclone, pVE807,
composed of the 2.56 kb Bg1II fragment inserted into the BamHI site of
pIJ922, complemented other mutants (Class II). Class III consists of the
mutants which are complemented by PVE650 but not by any subclones tested.
It is quite possible that each class may include more than one gene.
TABLE 2
__________________________________________________________________________
Complementation of S. avermitilis avermectin aglycone
producing mutants
Class
Mutants pVE650.sup.1
pVE908
pVE807
pVE941
pVE1018
pVE1420
pVE1116
__________________________________________________________________________
I GG900, MA6595,
+ + - - - - +
MA6586, MA6593,
MA6056, MA6624
II MA6582, GG898,
+ - + + - - +
MA6579, MA6581,
MA6589, MA6591,
MA5872
III MA6278, MA6580,
+ - - + - - +
MA6583, MA6584,
MA6585, MA6587,
MA6588, MA6060
IV MA6057, MA6622,
- - - + - - +
V MA6590 - - - + - + +
VI MA6592, MA6594
- - - + + + +
GMT MA6316, MA6323
- - - + - + +
__________________________________________________________________________
.sup.1 The indicated plasmid was transformed into the mutants and at leas
6 transformants were tested for avermectin production. The vector alone,
pIJ922, was indtroduced into the mutants and assayed as a negative
control.
Class I mutants have been tested by other subclones of pVE650. One other
subclone which complemented the mutants of Class I was the 4.05 kb EcoRI
fragment when cloned onto pIJ922 in one orientation (pVE808). However when
cloned in the other orientation (pVE818) a partially complemented or mixed
TLC pattern is observed which includes both aglycones and glycosylated
forms of avermectin. This may be the result of recombination correcting
the defect rather than complementation. Alternatively, the gene which
complements the defect in the Class I mutants may be poorly expressed from
pVE818 and there may be insufficient amounts of the protein necessary to
produce fully glycosylated avermectins. If this is so, it may indicate
that the promoter for these genes is located between the Bg1II site at
17.75 kb and EcoRI site at 19.67 kb on the pVE650 map. When the 2.36 kb
EcoRI-Bg1II fragment is cloned onto pIJ922, resulting in pIJ908, the Class
I mutants were also complemented, indicating that all the information to
correct the defects in these mutants is located within this fragment. The
gene represented by Class I is designated avrC.
Class II mutants are corrected by pVE807 which is pIJ922 with the 2.56 kb
Bg1II fragment. The gene represented by Class II mutants is designated
avrD.
Class III mutants are only complemented by pVE650. No complementation was
seen with either pVE808, pVE806, pVE807 or pVE845. pVE845 is a derivative
of pIJ922 with the 19.67 kb EcoRI fragment of pVE650 (FIG. 2) inserted in
the EcoRI site. pVE845, pVE806, pVE807, and pVE808 contain all the
sequences on pVE650. Since none of these clones complements Class III
mutants, the gene or operon represented by Class III mutants must span the
region of overlap by these plasmids. The gene represented by Class III
mutants is designated avrB.
Five aglycone producing mutants are not complemented by pVE650. Since a
cluster of at least 3 genes for oleandrose synthesis or addition was
located on pVE650, and this cluster was mapped to one end of the insert
DNA of pVE650, it was possible that the other gene(s) for oleandrose
synthesis or attachment were chromosomally located adjacent to the insert
on pVE650. As described below, cosmid clones containing this region were
isolated and a Bg1II fragment was identified which includes the region of
pVE650 beginning at the Bg1II site at 22.03 kb on the pVE650 map and
extending about an additional 14 kb. This Bg1II fragment was subcloned
onto pIJ922 to yield plasmid pVE941. This plasmid was found to complement
the aglycone producing mutants not complemented by pVE650. In addition,
pVE941 complemented GMT.sup.- strain, MA6316, indicating that the gene for
TDP-demethyl-oleandrose 3-O-methyltransferase, designated avrF, is also on
this fragment.
Southern hybridization analysis of pVE650 restriction fragments and genomic
DNA suggests that the insert DNA in pVE650 is colinear with the chromosome
and that there are two groups of reiterated or related sequences within
the insert. Probes made from three of the 10 BamHI fragments of pVE650
(the 2.22 kb, 1.09 kb, and 0.53 kb BamHI fragments) hybridize with only a
single fragment either in the chromosome or in pVE650. Group 1 consists of
1 BamHI fragment in pVE650 (2.09 kb), 1 fragment in pAT1 (0.55 kb BamHI),
and 5 other chromosomal fragments. Probes made from BamHI fragments in a
second group of related sequences, Group 2, hybridize with themselves as
well as 4 other BamHI fragments within pVE650 (the 7.0 kb, 4.6 kb, 3.0 kb,
1.82 kb, and 1.38 kb BamHI fragments) and 9 other chromosomal BamHI
fragments including one BamHI fragment in pAT1 (the 2.1 kb BamHI
fragment). The degree of homology, as indicated by the relative intensity
of hybridizing bands, varied significantly depending upon which of the
cloned fragments within each group was the probe suggesting inexact
sequence homology among these related sequences.
Isolation of cosmids containing a 110 kb avermectin gene cluster
As described above, 5 aglycone producing mutants are not complemented by
pVE650. Since the complementing region of pVE650 was located to one end of
the clone, it was possible other avermectin genes are located on the
adjacent chromosomal DNA. The 1.09 kb BamHI fragment of pVE650 (Table 4)
was used to probe a cosmid library of S. avermitilis DNA. DNA from 7 of
these clones which overlapped the 1.09 kb BamHI fragment were mapped with
respect to the different BamHI fragments of pVE650. One cosmid, pVE855,
contains all of the DNA on pVE650 and adjacent DNA on both sides. Another
cosmid, pVE859, extends at least 26 kb past the region of pVE650 that
complements the aglycone mutants (away from avrA the gene for avermectin
O-methyltransferase). At least 31 kb of DNA adjacent to pVE650 was cloned.
Since genes for antibiotic synthesis in Streptomyces are often clustered,
additional cosmid clones were isolated using the 2.09 kb BamHI fragment
(Table 4) from pVE650 to probe the S. avermitilis cosmid library. One
cosmid, pVE924, spans the 24 kb of DNA between the avrA clone, pAT1, and
pVE650. Thus, the cloned avermectin genes, avrA and the aglycone genes
avrB, avrC and avrD, define an avermectin gene cluster spanning over 55
kb. Another cosmid, pVE923, extends past the avrA region, away from the
aglycone region, about 20 kb. Collectively, over 110 kb of DNA has been
isolated from the avermectin gene cluster region. These plasmids were
mapped relative to each other by determining which BamHI fragments were
contained in common to one or more plasmids, by Southern analysis to
determine which plasmids contained BamHI fragments of the Group 1 and
Group 2 sets of related sequences, and via Southern analysis to test for
the presence on the plasmids of several fragments from the various
plasmids. The relative location of pAT1 , pVE650, and four cosmid clones
is indicated in FIG. 3.
pVE859 contains 6 Bg1II fragments of approximately 0.9 kb, 1.8 kb. 4.7 kb,
5.4 kb, 14 kb, and 18 kb. The 14 kb fragment was cloned into the unique
Bg1II site of pVE616, a 4.2 kb Amp.sup.r Thio.sup.r derivative of pBR322
with unique BamHi, BgIII, PstI and HpaI cloning site. pVE616 is incapable
of replicating in Streptomyces, but if it contains homologous DNA it can
integrate into the genome by recombination resulting in Thio.sup.r
derivatives. A derivative which contained the Bg1II fragment, pVE930, was
digested with a mixture of Bg1II and EcoRI restriction enzymes and
compared to pVE650 DNA digested with the same enzyme mixture. After
separation on agarose gels and visualization by UV illumination it was
observed that pVE650 and pVE930 contained a comigrating 1.55 kb
Bg1II-EcoRI fragment. This establishes that in the genome of S.
avermitilis the 14 kb Bg1II fragment cloned on pVE859 is adjacent to the
0.14 kb Bg1II fragment cloned on pVE650. The 14 kb Bg1II fragment was
subsequently subcloned into the BamHI site of pIJ922 to yield pVE941. This
plasmid was transformed into the aglycone producing mutants not
complemented by pVE650 and complementation was observed. In addition,
MA6316, a GMT.sup.- mutant was complemented by pVE941, the gene altered in
the GMT.sup.- strain is designated avrF. Thus, all the tested aglycone
mutants can be complemented by DNA on pVE650 and on pVE941, which
collectively contain about 37 kb of S. avermitilis DNA.
A 12 kb PstI fragment from pVE859 has been subcloned onto pVE1043 at a
unique PstI site, creating pVE1116. pVE1043 is a derivative of pIJ922 in
which the region from EcoRI to BamHI has been replaced with a poly linker
with unique sites for EcoRI, HpaI, PstI, NheI, AseI, HindIII, DraI, and
BamHI. Plasmid pVE1116, containing the 12 kb PstI fragment, was introduced
into mutants of all aglycone producing classes and the GMT mutants. As
indicated in Table 2, biosynthesis of natural avermectins was observed in
all mutants tested. Since this plasmid complements all mutants altered in
glycosylation of avermectin, it presumably contains all the genes for
glycosylation of avermectin. Additional restriction fragments were
subcloned onto pVE1043 and introduced into the mutants for complementation
studies. pVE1018 contains the 4.0 kb BamHI fragment from pVE941 and
pVE1420 contains the 3.8 kb PstI-EcoRI fragment from pVE1116. The results
are shown in Table 2. The defects in mutants MA6057 and MA6622 are
assigned to avrE, the defects in MA6592 and MA6594 are designated avrG,
and the defect in MA6590 is designated avrH. It is quite possible that
some complementation classes will be found to contain more than one gene.
FIG. 6 shows a restriction map of parts of pVE650, pVE941, and pVE1116 and
the location of the 7 avermectin genes involved in glycosylation.
Interestingly, pVE923, which was isolated with the 2.09 kb BamHI probe,
does not contain DNA that overlaps the 2.09 kb BamHI fragment probe.
Plasmid pVE923 was isolated because it contains two other related
sequences of Group 1. The four plasmids pVE923, pVE924, pVE855 and pVE859
have been inserted into strains of Escherichia coli using standard
techniques and the cultures deposited to ensure availability. The four E.
coli strains containing the 4 cosmid clones have been designated MB5373
(pVE923) deposited as ATCC 67891; MB5374 (pVE924) deposited as ATCC 67892;
MB5370 (pVE855) deposited as ATCC 67889; and MB5372 (pVE859) deposited as
ATCC 67890. The sizes of the BamHI fragments in these 4 cosmids, as well
as pAT1 and pVE650, have been determined and are presented in Table 5.
Mapping cosmid pVE924 by constructing subclones of pVE924
Cosmid pVE924 spanned the region from pVE650 to pAT1. Since genes for
antibiotic biosynthesis are often clustered, it was possible that other
avermectin genes would be linked to the genes for O-methylation and
glycosylation. The test this hypothesis the BamHI fragments form pVE924
were subcloned onto pVE616, pVE1053 (a derivative of pVE616), or pVE623 (a
derivative of pIJ922). pVE924 was partially digested with BamHI and cloned
into the uniqued BamHI site of the above three vectors. A set of clones
containing 1 or more BamHI fragments was isolated. From the clones with
more than one BamHI fragment, a map of the relative order of BamHI
fragments was determined. FIG. 5 displays the restriction map of the BamHI
fragments from pVE924.
Isolation of genes involved in synthesis of the avermectin macrocyclic
lactone ring
Five subclones of pVE924 (indicated by an * in FIG. 5) which collectively
represent the DNA of pVE924, as well as plasmids pAT1, pVE650 and pVE941
were used in complementation experiments with 24 avermectin non producers
(Avr), two C-22, C-23 dehydrase (DH) mutants, and a mutant unable to close
the avermectin furan ring (FUR). Twelve mutants were complemented,
including the DH, FUR and 9 Avr mutants. These mutants formed 8
complementation classes. The DH mutants represent avrI and FUR mutant
represents avrJ. The 6 classes of nonproducers represent avrK, avrL, avrM,
avrN, avrO, and avrP. The location of these genes is indicated on FIG. 3.
These results clearly show the DNA cloned on pVE923, pAT1, pVE924, pVE855,
pVE650 and pVE859 contain many avermectin genes. Subcloning of all the DNA
from this avermectin gene cluster will allow identification of the genes
for avermectin biosynthesis.
Isolation of additional DNA from the avermectin gene cluster
A comparison of the restriction maps of pAT1, pVE923 and pVE924 showed that
the region adjacent to the 0.55 kb BamHI fragment was different in the
three clones. On pAT1, a 3.4 kb BamHI to vector juction fragment, which
contains an EcoRI site, maps adjacent to the 0.55 kb BamHI fragment. On
pVE924 a 3.2 kb BamHI fragment without an EcoRI site is located adjacent
to the 0.55 kb BamHI fragment. Cosmid pVE923 has a 7.0 kb BamHI fragment
located adjacent to the 0.55 kb BamHI fragment. In order to determined the
actual structure of this region of the avermectin gene cluster, DNA from
the S. avermitilis chromosome was directly cloned into E. coli.
This method relied on the homologous recombination system of S. avermitilis
to direct the integration of an E. coli plasmid containing two fragments
of the avermectin cluster which flank the region of interest. This
plasmid, pVE1299, is a derivative of pVE616 (Thio.sup.r) which contains
the 3.4 kb BamHI fragment of pAT1, a 2.9 kb neomycin-resistance gene (neo)
fragment form Tn5, and the 3.7 kb BamHI fragment from pVE924. The vector
can not replicate in S. avermitilis. Upon transformation into S.
avermitilis, transformants containing the plasmid integrated in the
chromosome were isolated as Thio.sup.r, Neo.sup.r. After excision of this
vector from S. avermitilis by recombination, the resulting plasmid DNA was
isolated and used in transformation of E. coli. Plasmids were recovered in
which the neo DNA was replaced with S. avermitilis DNA that is located
between the two fragments originally cloned on pVE1299. Restriction
mapping of one such plasmid, pVE1446, revealed that there were actually
three chromosomal BamHI fragments (7.0 kb, 7.4 kb, and 8.0 kb) between the
0.55 and 3.7 kb BamHI fragments. Table 6 presents a restriction map of
over 95 kb of DNA from the avermectin gene cluster region.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1. Restriction map of pAT1. Only the sites mapped in both the vector
and insert DNA are indicated.
FIG. 2. Restriction map of pVE650. Only the sites mapped in both the vector
and insert DNA are indicated.
FIG. 3. A map of the avermectin gene cluster. The extent of DNA contained
on pVE923, pAT1, pVE1446, pVE924, pVE855, pVE650, pVE1116, and pVE859 is
indicated. The deleted region on pVE924 is indicated by a hollow line.
Regions which complement avermectin mutants are indicated. avrA is the
gene for avermectin C-5 O-methyltransferase. avrB, C, D, E, G, and H
represent genes defective in the Class III, Class I, Class II, Class IV,
Class VI, and Class V, aglycone producing mutants. avrF is the gene for
avermectin glycosyl O-methyltransferase. Avermectin C-22, C-23 dehydrase
is designated avrI, the gene involved in forming the furan ring in
avermectin is designated avrJ, and six genes involved informing the
macrocyclic lactone ring are designated avrK, avrL, avrM, avrN, avrO and
avrP. The approximate locations of regions of related sequences in Group 1
and Group 2 are indicated.
FIG. 4. Restriction map of pVE328.
FIG. 5. Restriction map of the insert in cosmid pVE924 and BamHI partial
subclones. Vertical lines represent BamHI sites. Numbers 2 to 15 represent
the second largest fragment to the smallest BamHI fragments of pVE924
listed in Table 5. Plasmids indicated with an * were used in
complementation tests.
FIG. 6. A restriction map of the glycosylation region. The BamHI site
indicated by a dotted line is present on pVE650 at the insert/vector
junction but not in the chromosome. The zero kb origin represents the
beginning of the insert in pVE650. The extent of DNA on pVE650, pVE1116,
pVE941, pVE908, pVE806, pVE807, pVE808, pVE1018, and pVE1420 is indicated.
The locations of regions which complement mutants defective in
glycosylation of avermectin are indicated.
FIG. 7. Restriction map of pVE1011. The ** mark the cloning sites used to
form pVE1299.
This application describes the successful cloning of avermectin genes using
low copy number vectors to complement S. avermitilis mutants blocked in
avermectin biosynthesis. In U.S. Pat. No. 4,703,009 an example of how to
clone genes for avermectin biosynthesis in a high copy number vector was
described but this description is flawed. First such described high copy
number vectors will not replicate successfully with large fragments.
Second such high copy number vectors which contain S. avermitilis inserts
apparently undergo recombination with the genome. These plasmids are
difficult to isolate and characterize. Third high copy number clones alter
the regulation of avermectin genes which makes detection of complementing
clones difficult.
UTILITY
The avermectin gene cluster region from S. avermitilis, which has been
cloned on several plasmids, can be used to create new processes to produce
avermectins, can be used to produce new avermectins, and can be used to
create hybrid antibiotics. Several of the genes required for avermectin
biosynthesis have been localized by subcloning. Other avermectin genes are
located within the region cloned here and/or near the region cloned here.
There are many uses for the cloned S. avermitilis DNA.
Cloned avermectin biosynthesis genes may be used to alter the normal
composition of the 8 natural product avermectins, resulting in new
processes. Introduction of the C-5 O-methyltransferase gene, which has
been subcloned from the avermectin gene cluster region, into S.
avermitilis strains, results in the enhanced production of C-5 methoxy
components. Similarly, the gene for avermectin C-22, C-23 dehydrase can be
introduced into S. avermitilis strains to enhance the production of
dehydrated components.
Identification, from the region cloned here, of a gene encoding an enzyme
which is a rate limiting step in avermectin biosynthesis, can be used to
create an improved process for avermectin production. This would occur by
subcloning the gene and manipulating it to increase the expression of the
limiting enzyme.
Novel avermectins can be produced by mutagenesis of the cloned genes.
Mutagenesis of the glycosylation genes can yield strains which produce
predominately monosacharide containing avermectins. Mutagenesis of the
genes for the synthesis of the avermectin aglycone can result in novel
avermectins. Avermectin is synthesized by the sequential addition of short
chain carboxylic acids which may retain a keto group or the keto group may
be reduced to a different functional group such as a hydroxyl, a double
bond or a saturated bond. Mutagenesis of the cloned avermectin genes can
result in the synthesis of avermectin with a different carboxylic acid
than the natural avermectins, or avermectins with different
functionalities.
The DNA from the avermectin gene cluster region can be used as a
hybridization probe to identify homologous sequences. Thus, the DNA cloned
here could be used to locate additional plasmids from the S. avermitilis
gene libraries which overlap the region described here but also contain
previously uncloned DNA from adjacent regions in the genome of S.
avermitilis. In addition, DNA from the region cloned here may be used to
identify non-identical but similar sequences in other organisms. The genes
for avermectin glycosylation can be used to identify and isolate genes
involved in glycosylation of other antibiotics. The gene for C-5
avermectin O-methyltransferase can be used as a DNA probe to identify
other O-methyltransferase genes and aid in their cloning. The introduction
of these heterologous genes into S. avermitilis can result in potent
anthelmintics by the synthesis of novel avermectins with altered
methylation or glycosylation. Similarly, DNA containing genes for the
synthesis of the avermectin aglycone can be used as a probe to identify,
and subsequently clone, sequences for similar macrolides. The genes for
other anthelmintics such as milbemycins could also be cloned in this way.
The complementation S. avermitilis mutants with the appropriate genes from
the milbemycin producing strains can result in potent, hybrid anthelmintic
compounds.
The following examples are provided in order that the invention might be
more completely understood. They should not be construed as limitation of
the invention.
EXAMPLE 1
Isolation, Maintenance and Propagation of Plasmids
The plasmid DNA was isolated and handled by procedures differing little
from those established by work on other plasmids. A good procedures manual
is T. Maniatis, E. F. Fritsch, and J. Sambrook, Molecular Cloning: a
Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
(1982). A good procedures manual for Streptomyces is D. A. Hopwood, M. J.
Bibb, H. F. Chater, T. Kieser, C. J. Bruton, H. M. Kieser, D. J., Lydiate,
C. P. Smith, J. M. Ward, and H. Schrempf, Genetic Manipulation of
Streptomyces, a Laboratory Manual, John Innes Foundation, Norwich, UK
(1985). The specific procedures used in this work are described herein,
unless they are identical to those given in the above mentioned manuals.
A. Growth of Streptomyces for Plasmid Isolation.
Single colonies of Streptomyces strains were isolated on R2YE, medium A or
medium D. R2YE containing 103 g sucrose, 10 g glucose, 3 g yeast extract,
3 g proline, 0.1 g casamino acids, 0.25 g K.sub.2 SO.sub.4, 10.1 g
MgCl.sub.2.6H.sub.2 O, 2 ml of R2 trace elements (0.2 g
FeCl.sub.3.6H.sub.2 O, 0.04 g ZnCl.sub.2, 0.01 g MnCl.sub.2.4H.sub.2 O,
0.01 g CuCl.sub.2.2H.sub.2 O, 0.01 g NaB.sub.4 O.sub.7.10H.sub.2 O, and
0.1 g (NH.sub.4).sub.6 Mo.sub.7 O.sub.24.4H.sub.2 O per liter) and 15 g
agar brought to a final volume of 940 ml with distilled water. After
autoclaving, the following solutions were added to each liter of medium:
10 ml of 0.5% NH.sub.4 PO.sub.4, 8 ml of 2.5M CaCl.sub.2, and 40 ml of
0.2M TES pH 7.2 (N-tris-(hydroxyethyl) methyl-2-amino ethanesulfonic
acid). Medium D contained 4 g yeast extract, 10 g malt extract, 4 g
glucose, 5 ml of trace elements (50 ml of 37.3% HCl, 61.1 g
MgSO.sub.4.7H.sub.2 O, 2.0 g CaCO.sub.3, 5.4 g FeCl.sub.3.6H.sub.2 O, 1.44
g ZnSO.sub.4.7H.sub.2 O, 1.11 g MnSO.sub.4.H.sub.2 O, 0.25 g
CuSO.sub.4.5H.sub.2 O, 0.062 g H.sub.3 BO.sub.3 and 0.49 g Na.sub.2
MoO.sub.4.2H.sub.2 O per liter), and 15 g agar per liter. The pH of medium
D was adjusted to pH 7.0 with NaOH before autoclaving. Liquid growth
medium for Streptomyces was YEME (3 g yeast extract, 5 g peptone, 3 g malt
extract, 10 g glucose per liter) modified to contain 30% sucrose, 5 mM
MgCl.sub.2 and included antibiotics to select for the maintenance of
plasmids. Strains with plasmids containing a thiostrepton-resistance gene
(tsr) were grown with 5 mg per ml of thiostrepton. A single colony was
inoculated into 6 ml of YEME in a 10 mm by 150 mm tube. The culture was
grown for 3 days at 28.degree. C. with shaking at 220 rpm.
(1) Small scale plasmid isolation.
For small scale plasmid preparations, mycelia from the 6 ml YEME culture
were collected by centrifugation at 14,000.times.g for 12 minutes. The
pellet was washed once in 10% sucrose, 10 mM ethylenediamine tetraacetate
(EDTA), pH 8.0. Plasmid DNA was isolated from the mycelia by a rapid
boiling procedure described previously by MacNeil, D. J., J. of Microbiol.
Methods pg 115-123, (1986). The pellet was resuspended in 0.5 ml of STET
(8% sucrose, 5% Triton X-100, 50 mM EDTA and 50 mM Tris, pH 8.0), 30 .mu.l
of a 30 mg/ml lysozyme (Sigma, St. Louis, Ma.) solution was added, the
mixture was incubated for 15 minutes at 37.degree. C., and then placed in
a boiling water bath for 2 minutes. The boiled lysate was spun at
14,000.times.g for 12 minutes, the supernatant was removed to a 1.5 ml
Eppendorf tube, and then extracted once with phenol previously
equilibrated with TE (10 mM Tris, 1 mM EDTA, pH 7.9). The aqueous phase
was removed to another 1.5 ml Eppendorf tube, an equal volume of
isopropanol was added, the solutions were incubated at -20.degree. C. for
20 minutes, and the DNA was pelleted at 7000.times.g for 6 minutes. After
washing once in 70% ethanol, the DNA was resuspended in 100 .mu.l of TE.
An estimated 2 to 10 .mu.g of plasmid DNA was obtained from a 6 ml
culture. Alternatively, plasmid DNA was isolated from 6 ml cell pellet by
an alkaline lysis procedure. The cell pellet was resuspended in 1 ml of 50
mM glucose, 25 mM Tris pH 8, 10 mM EDTA, and 50 .mu.l of a 30 mg/ml
lysozyme solution in 50 mM glucose, 25 mM Tris pH 8, 10 mM EDTA was added.
Following incubation for 15 minutes at 37.degree. C., 1.5 ml of a 0.2N
NaOH, 1% SDS solution was added, the mixture was vortexed for 5 seconds
and the mixture was incubated for 15 minutes on ice. Next 150 .mu.l of ice
cold pH 4.8 potassium acetate solution (5M with respect to acetate, 3M
with respect to potassium) was added, the mixture vortexed for 10 seconds,
and incubated on ice for 15 minutes. The mixture was centrifuged for 15
minutes at 12,000.times.g, at 4.degree. C. and the resulting supernatant
was transferred to a new tube. 2.5 ml of -20.degree. C. isopropanol or
isopropanol containing 0.05% diethyl pyrocarbonate was added, mixed, and
centrifuged at 12,000.times.g for 15 minutes at 4.degree. C. Remaining
solvent from the resulting DNA pellet was removed in a Savant Speed Vac,
and the DNA was dissolved in 0.5 ml of 0.3M ammonium acetate. The solution
was transferred to a 1.5 ml Eppendorf tube, mixed with 400 .mu.l of
phenol, previously equilibrated with 1M Tris pH 7.9, and the aqueous phase
separated by centrifugation in a microfuge for 3 minutes. The aqueous
phase was removed to another Eppendorf tube and the phenol extracted with
400 .mu.l of chloroform. The resulting aqueous DNA solution was
precipitated with 2 volumes of ethanol at -70.degree. C. for at least 20
minutes. The DNA was collected by centrifugation in a microfuge for 15
minutes, washed once with -20.degree. C. 70% ethanol, dryed in a Savant
Speed Vac, and resuspended in 100 .mu.l of TE buffer.
(2) Large Scale plasmid isolation.
For large scale plasmid isolations a 6 ml YEME culture was used to
inoculate a 250 ml baffled flask containing 30 ml of YEME. After 2 days of
shaking at 28.degree. C. at 220 rpm the culture was used to inoculate a
baffled 2 liter flask containing 500 ml of YEME. The mycelia were
harvested by centrifugation at 4,000.times.g for 15 minutes and were
washed once in 10% sucrose, 10 mM EDTA. Plasmid DNA was isolated from the
mycelia by either of two methods. One method was a rapid boiling procedure
as described previously by MacNeil, D. J., 1986, supra. The cell pellet
was resuspended in 40 ml of STET, and 0.5 ml of 50 mg/ml lysozyme solution
in 0.1M Tris pH 7.9 was added. The suspension was incubated at 37.degree.
C. for 20 minutes, placed in a boiling water bath for 3 minutes and
centrifuged at 90,000.times.g for 30 minutes at 4.degree. C. The
supernatant was removed, one half volume of -20.degree. C. isopropanol was
added, mixed and incubated at -20.degree. C. for 20 minutes. DNA was
collected by centrifugation at 9,000.times.g for 8 minutes. The DNA was
resuspended in 13 ml of a CsCl solution prepared by dissolving 78 g of
CsCl into 65 ml of 0.1M Tris, 0.01M EDTA, pH 7.9 and adding 2 ml of
ethidium bromide (5 mg/ml). The mixture was centrifuged at 43,000 rpm in
a Beckman ultracentrifuge for 44 hours. The second method to isolate
plasmid DNA was a modification of the alkaline lysis procedure described
by Maniatis et al., 1982, supra. The 500 ml cell pellet was resuspended in
30 ml of 50 mM glucose, 25 mM Tris pH 8, 10 mM EDTA and 2 ml of 15 mg/ml
lysozyme solution was added. The mixture was swirled occasionally during
incubation at 37.degree. C. for 30 minutes. 50 ml of 0.2N NaOH, 1% SDS,
was added and the mixture was mixed with a 1 ml pipet until the mixture
appeared homogeneous and lysis was evident. After incubation on ice for 25
minutes with occasional swirling, 40 ml of 5M potassium acetate pH 4.8 was
added and mixed until the precipitated material was dispersed into small
clumps. After incubation on ice for 25 minutes, the mixture was
centrifuged at 15,000.times.g for 15 minutes at 4.degree. C. The plasmid
containing supernatant was added to 72 ml of -20.degree. C. isopropanol
mixed and centrifuged at 15,000.times.g for 15 minutes at 4.degree. C. The
resulting supernatant was discarded, excess liquid was removed with a
sterile cotton swab and the DNA pellet dryed further under vacuum for 5
minutes. The DNA was resuspended in 9 ml of 20 mM Tris, 0.5% sarkosyl, 5
mM EDTA, pH 7.9 plus 25 .mu.l of 10 mg/ml RNase, the volume was brought up
to 10 ml, 11 g of CsCl was added and 1 ml of a 5 mg/ml solution of
ethidium bromide was added. After centrifugation at 5000.times.g for 5
minutes the supernatant was added to a Beckman Quick Seal tube, sealed and
spun at 65,000 RPM in Beckman 70.1 Ti rotor for 17.5 hours at 20.degree.
C. The plasmid DNA band, obtained from either method, was visualized by UV
illumination, was removed and rebanded in 13 ml of a CsCl solution
prepared by dissolving 71 g of CsCl into 65 ml of 0.1M Tris, 0.01M EDTA,
pH 7.9 and adding 0.2 ml of ethidium bromide (5 mg/ml). The plasmid DNA
was removed from the second gradient and ethidium bromide was removed by 4
isopentyl alcohol extractions. The plasmid DNA was precipitated by adding
2 volumes of TE, 0.3 volumes of 3.5M sodium acetate, and 6 volumes of 100%
ethanol. After overnight incubation at -20.degree. C. the DNA was pelleted
by centrifugation at 13,000.times.g for 12 minutes, washed once with 70%
ethanol, and resuspended in 1 ml of TE. The yield of DNA from 500 ml of
cells was 200 to 1000 .mu.g.
B. Growth of E. coli for plasmid isolation.
E. coli cultures containing pVE328-derived cosmid clones were grown in
LB-Amp medium (10 g tryptone, 5 g yeast extract, 5 g, NaCl per liter
containing 100 .mu.g/ml of ampicillin). These cultures were grown at
37.degree. C. shaking at 220 rpm when the OD.sub.600 was between 1.0 to
2.0, 0.5 ml of 50 mg/ml chloramphenicol was added. Incubation continued
overnight at 37.degree. C. Large amounts of plasmid DNA (200 to 1,500
.mu.g) were prepared from a 500 ml culture by a modification of the
alkaline lysis procedure described above for Streptomyces. Cells were
collected at 6,000.times.g for 6 minutes, the cell pellet was resuspended
in 18 ml of 50 mM glucose, 25 mM Tris pH 8, 10 mM EDTA and 2 ml of 15
mg/ml lysozyme solution added. The mixture was swirled occasionally during
incubation at room temperature for 15 minutes. Forty ml of 0.2N NaOH, 1%
SDS, was added and the mixture was mixed with a 1 ml pipet until the
mixture appeared homogeneous and lysis was evident. After incubation on
ice for 25 minutes with occasional swirling, 20 ml of 5 M potassium
acetate pH 4.8 was added and mixed until the precipitated material was
dispersed into small clumps. After incubation on ice for 25 minutes the
mixture is centrifuged at 15,000.times.g for 15 minutes at 4.degree. C.
The plasmid containing supernatant was added to 50 ml of -20.degree. C.
isopropanol, mixed, and centrifuged at 15,000.times.g for 15 minutes at
4.degree. C. The resulting supernatant was discarded, excess liquid was
removed with a sterile cotton swab, and the DNA pellet dried further under
vacuum for 5 minutes. The DNA was resuspended in 9 ml of 20 mM Tris, 5 mM
EDTA, pH 7.9 plus 25 .mu.l of 10 mg/ml RNase, the volume was brought to 10
ml, 11 g of CsCl was added and 1 ml of a 5 mg/ml solution of ethidium
bromide was added. After centrifugation at 5000.times.g for 5 minutes, the
supernatant was added to a Beckman Quick Seal tube, sealed, and spun at
65,000 RPM in Beckman 70.1 Ti rotor for 17.5 hours at 20.degree. C. The
plasmid DNA band was visualized by UV illumination, removed, and rebanded
in 13 ml of a CsCl solution prepared by dissolving 71 g of CsCl into 65 ml
of 0.1M Tris, 0.01M EDTA, pH 7.9 and adding 2 ml of ethidium bromide (5
mg/ml). The plasmid DNA was removed from the second gradient and ethidium
bromide was removed by 4 isopentyl alcohol extractions. The plasmid DNA
was precipitated by adding 2 volumes of TE, 0.3 volumes of 3.5M sodium
acetate, and 6 volumes of 100% ethanol. After overnight incubation at
-20.degree. C., the DNA was pelleted by centrifugation at 13,000.times.g
for 12 minutes, washed once with 70% ethanol and resuspended in 1 ml of
TE.
C. Restriction analysis of plasmid DNA.
Procedures for restriction analysis of DNA and agarose gel electrophoresis
as well as other standard techniques of recombinant DNA technology are
thoroughly described in T. Maniatis, E. F. Fritsch, and J. Sambrook,
Molecular Cloning: a Laboratory, Manual, Cold Spring Harbor Laboratory,
Cold Spring Harbor, N.Y. (1982). Plasmid DNA isolated from large and small
scale preparations was cleaved with various restriction enzymes according
to the manufacture's directions. Enzymes were obtained from New England
Biolabs (Beverly, Mass.), Bethesda Research Labs (Bethesda, Md.), and IBI
(New Haven, Conn.). The digestions were analyzed by electrophoresis in
0.8% agarose using 0.08M Tris-acetate-0.004M EDTA as a buffer. The size of
the fragments was determined by comparison to fragments of phage lambda
DNA of known molecular weight.
D. Mapping restriction enzyme sites in cloned DNA.
The location of restriction sites in pAT1 and pVE650 was determined by
standard mapping techniques. These included analysis of single and double
enzyme digestions, by subcloning and analysis of the subclones, and by
analysis of the fragments contained on various cosmids isolated from the
region. The size of the fragments was determined by comparison to lambda
fragments digested with HindIII or a combination of BamHI and EcoRI.
E. Transformation of Streptomyces by plasmid DNA.
(1) Protoplast formation.
Transformation was accomplished by PEG-mediatd DNA uptake by protoplasts.
Protoplasts of S. avermetilis were prepared as described by MacNeil, D. J.
and Klapko, L. M., 1987, J. Indust. Microbiol. 2: 209-218. Thirty ml of
YEME medium containing 30% sucrose, 5 mM MgCl.sub.2 and 0.5% glycine was
inoculated with 5.times.10.sup.7 spores of S. avermitilis, or with 1 ml of
6 ml YEME culture. (The YEME culture was prepared by inoculating a single
colony into 6 ml of YEME containing 30% sucrose, 5 mM MgCl.sub.2, and 0.5%
glycine). The 30 ml culture was grown for 2 or 3 days at 28.degree. C.,
the mycelia were pelleted at 14,00.times.g for 12 minutes and washed once
with P medium. P medium contains 103 g sucrose, 0.25 g K.sub.2 SO.sub.4,
2.03 g MgCl.sub.2.H.sub.2 O, and 2 ml of R2 trace elements per liter.
After autoclaving the following additions were made; 10 ml of 0.5%
KH.sub.2 PO.sub.4, 8 ml CaCl.sub.2.2H.sub.2 O, 40 ml of 0.2M MES
(2-(N-morpholino)ethanesulfonic acid). The mycelium was resuspended in 15
ml of P medium, 0.2 ml of lysozyme (50 mg/ml in P medium) was added, and
the suspension was incubated at 37.degree. C. for 1 hour with slow
shaking. Protoplasts were separated from undigested mycelium by filtering
the mixture through 2 cm of glass wool in the bottom of a 10 ml syringe.
The protoplasts were pelleted at 6,000.times.g for 6 minutes and
resuspended in 3 ml of P medium which contained 20% sucrose. Protoplasts
of S. lividans were prepared as described above for S. avermitilis, except
TES (pH 7.2) was used in all buffers instead of MES. All protoplasts were
quick frozen in a dry ice/ethanol bath and stored at -70.degree. C.
(2) Transformation procedure.
Streptomyces strains were transformed by modification of the method
described for S. avermitilis (MacNeil and Klapko, 1987, supra). A quantity
of 0.1 ml of protoplasts (approximately 10.sup.9 /ml) were mixed with 5-10
.mu.l of plasmid DNA (25 ng-1 .mu.g), 0.5 ml of medium T was added and the
mixture incubated for 30 seconds at room temperature. On some occasions
half as much protoplasts, DNA, and medium T were mixed together. Medium T
is similar to medium P except it contains different concentrations of
sucrose (2.5%), CaCl.sub.2 (0.1M) and is buffered with 50 mM Tis-maleic
acid (pH 8) and has 25% (wt.vol) PEG 1000. The mixture was serially
diluted in P medium containing either 0.01M MES for S. avermitilis or
0.01M TES for S. lividans. For S. lividans, 100 .mu.l of the dilutions of
the transformation mixtures were spread on R2YE medium containing 17%
sucrose. For S. avermitilis 100 .mu.l of the dilutions of the
transformation mixtures were added to 3 ml of RM14 soft agar at 50.degree.
C. and poured onto RM14 plates. RM14 is similar to R2YE except it contains
205 g sucrose, 20 g agar, 3 g of oatmeal agar per liter, and 0.1M MES (pH
6.5) instead of TES. RM14 soft agar contains only 6 g of agar per liter.
(3) Detection of transformation.
R2YE and RM14 regeneration media containing the transformed protoplasts
were incubated for 20 hours at 28.degree. C. Regeneration plates were
overlayed with 3 ml of RM14 soft agar containing 0.5 mg of thiostrepton.
Transformants appeared on the regeneration plates in 4 to 15 days.
E. Transformation of E. coli by plasmid DNA.
Competent cells of E. coli were prepared by the method of Mandel, M. and
Higa, A., J. Mol. Biol., 53 pg 154-162 (1970). Cells were grown in LB
medium to an A.sub.600 =0.45 and incubated on ice 20 minutes. Cells were
pelleted and resuspended to one-half their original volume in 0.1M
CaCl.sub.2. After 20 minutes on ice, cells were again pelleted and
resuspended to 0.1 of their original volume in 0.1M CaCl.sub.2. These
competent cells were made 15% glycerol and stored at -70.degree. C.
For transformations, 0.2 ml of competent cells was mixed with 10 .mu.l of
DNA (10 to 1000 ng/ml). The mixture was incubated on ice for 10 minutes
then at 37.degree. C. for 3 minutes. A quantity of 0.5 ml of LB medium was
added and the culture was shaken at 37.degree. C. at 220 RPM for 1 hour.
Aliquots were plated on LB plates with 100 .mu.g/ml of ampicillin to
select for the plasmids.
F. Subcloning fragments into pIJ922 for complementation tests
To aid in locating genes for avermectin biosynthesis DNA fragments from
pAT1, pVE650 and pVE859 were subcloned onto pIJ922. Two to ten micrograms
of pIJ922 were linearized at the unique BamHI or EcoRI sites in pIJ922 by
digestion with the appropriate restriction enzyme. The linear DNA was
treated with calf intestinal alkaline phosphatase (CIAP) (Boehringer
Mannheim, Indianapolis, Ind.) as described by the manufacturer or by
alternative procedures. A simple method was to treat the linearized pIJ922
immediately after the completion of the restriction enzyme digestion.
CIAP, (0.02 units per .mu.g of DNA) was added directly to the restriction
enzyme digestion mixture and incubated for 30 minutes at 37.degree. C. A
second aliquot of CIAP was added and the digestion continued for another
30 minutes. The reaction was terminated by the addition of 1/5 volume of
100 mM EDTA, 25% glycerol, 0.25% bromephenol blue, 0.2% SDS. The linear
vector was electrophoresed on a 0.8% agarose gel and the linear DNA was
electro-eluted from the agarose slice containing the DNA. The DNA was
ethanol precipitated and resuspended in 50 to 100 .mu.l of TE. For
subcloning, 5 to 10 .mu.g of pAT1, pVE650 or pVE859 were digested with a
restriction enzyme BamHI, Bg1II, PstI or, EcoRI, electrophoresed in a 0.8%
agarose gel, electroeluted, ethanol precipitated and resuspended in 50 to
100 .mu.l of TE. Various aliquots of the BamHI linearized vector and BamHI
or Bg1II digested fragments, or EcoRI linearized vector and EcoRI digested
fragment, or PstI linearized vector and PstI digested fragment, were
ligated and transformed into S. lividans. Thio.sup.r transformants were
selected and tested for the presence of the fragment of interest by
minilysate analysis of 6 ml YEME grown cultures. Derivatives with the
appropriate insert were saved and 5 to 10 .mu.l of the minilysate were
transformed into various S. avermitilis mutants, and the resulting
thiostrepton-resistant transformants tested for complementation of the
mutant defect.
EXAMPLE 2
Isolation and characterization of pATl
A. Construction of a S. avermitilis DNA library.
A library of S. avermitilis DNA was made by ligating genomic DNA partially
digested with Sau3A restriction enzyme into the compatible and unique
BamHI site of pIJ922. pIJ922 is a 24 kb, low copy number, Streptomyces
vector which can be selected as Thio.sup.r in Streptomyces (see Hopwood et
al., Genetic Manipulations of Streptomyces a Laboratory Manual, pg 279).
(1) Isolation of Genomic DNA.
An S. avermitilis spore stock was prepared by spreading 0.1 ml of a visibly
turbid YEME grown culture onto Medium D agar plates. After 5 to 15 days
the spores are removed with a cotton swab, wetted with 0.85% NaCl, 50%
glycerol and transferred to 2 ml of 0.85 NaCl, 50% glycerol. 100 .mu.l of
the S. avermitilis spore stock (ca. 5.times.10.sup.9 spore/ml) was
inoculated into a 250 ml baffled flask containing 30 mls of YEME+30%
sucrose+0.5% glycine. After 4 days incubation at 27.degree. C. on a rotary
shaker at 220 rpm, the cells were harvested by centrifugation at
12,000.times.g for 10 minutes at 4.degree. C. using a 25 ml Corex
centrifuge tube. The cell pellet was resuspended in 20 ml of P medium
containing 0.01M MES, pH 6.5, centrifuged at 12,000.times.g for 10
minutes at 4.degree. C., and the pellet was resuspended in 5 ml of P
medium, 10 ml of lysozyme was added and mixed. The suspension was
incubated at 37.degree. C. for 1 hour and then 2.5 ml of 0.25M EDTA, pH
8.0 was added and the incubation continued on ice for 15 minutes. Cell
lysis was evident after the addition of 7.5 ml of 2% sarkosyl by gently
swirling the mixture. Following an additional 30 minute incubation on ice,
150 .mu.l of a solution of preboiled, 5 mg/ml RNAse Type 1A (Sigma, St.
Louis, Mo.) was added and incubation continued for 1 hour at 37.degree. C.
Next, 0.6 ml of Proteinase K (IBI, New Haven, Conn.) was added and the
mixture was incubated for 2 hours at 37.degree. C., then incubated at
4.degree. C. overnight. (Proteinase K was predigested by incubating a 25
mg/ml solution at 37.degree. C. for 1 hour). Fifteen ml of phenol,
previously equilibrated with 0.2M Tris, pH 7.9, was added to the lysate
and mixed at 8 rpm on a rotating mixer (Rugged Rotator, Kraft Devices,
Mineola, N.Y.). After spinning the mixture in a table top centrifuge, the
aqueous layer was transferred to a new tube and the extraction repeated 2
more times. Phenol was removed from the sample by 5 extractions with
chloroform:isoamyl alcohol, 24:1. To the resulting aqueous solution, 0.5
volumes of 7.5M ammonium acetate were added, mixed, and 2 volumes of
ice-cold ethanol were added. Using a glass rod, the ethanol was mixed and
the resulting precipitated DNA spooled on to the rod. Excess solution was
removed from the DNA by pressing the rod against the side of the tube, and
the DNA was dissolved in 5 ml of TE containing 4 .mu.l of diethyl
pyrocarbonate. The DNA was dissolved overnight while rotating at 8 rpm on
the rotating mixer. The DNA was then again precipitated from the solution
as described above and finally dissolved in 2.0 ml of TE to yield 469
.mu.g/ml of DNA as determined by the OD.sub.260 measurement.
(2) Size fractionation of the genomic DNA.
After a pilot experiment to determine conditions to dilute MboI and Sau3A
to yield a maximum of fragments in the 20 to 40 kb range, four aliquots of
100 mg of S. avermitilis DNA were digested at 37.degree. C. with 10 U,
16.7 U, 23.3 U, and 30 U of MboI and four aliquots of 100 .mu.g of S.
avermitilis DNA were digested at 37.degree. C. with 3 U, 5 U, 7 U, and 9 U
of Sau3A in a 1 ml reaction mixture for 10 minutes. The same buffer, KCl
Buffer, was used for both enzymes and it was 20 mM Tris, 20 mM KCL, 10 mM
MgCl.sub.2, 1 mM dithiothreitol, 100 .mu.g/ml bovine serum albumin, pH
7.9. The reactions were terminated by addition of 0.25 ml of stop mix (1%
SDS, 50 mM EDTA) followed by phenol extraction. Unless otherwise
indicated, DNA solutions were treated with phenol to inactivate enzymes
and remove protein. The standard phenol extraction consists of sequential
extractions of the DNA solution with organic solvents followed by
centrifugation to separate the phases. The extractions are done with equal
volumes of phenol, previously equilibrated with 0.2M Tris pH 7.9;
phenol:chloroform:isoamyl alcohol 25:24:1; chloroform:isoamyl alcohol
24:1. The DNA may be concentrated and/or the salts removed by ethanol
precipitation. The standard ethanol precipitation of DNA consists of the
addition of 0.5 volume 7.5M ammonium acetate, 3 volumes of ethanol, and
incubation at -20.degree. C. overnight or -70.degree. C. for 1 hour. The
DNA is pelleted by centrifugation at 12,000.times.g for 15 minutes, washed
with 70% ethanol, dryed, and resuspended in TE. Following phenol
extraction and ethanol precipitation, the MboI digestions were pooled and
the Sau3A digestions were seperately pooled and loaded on a 32 ml 10% to
40% sucrose gradient (1M NaCl, 20 mM Tris, 5 mM EDTA, pH 8.0). After 21
hours of centrifugation in a Beckman SW 28 rotor at 26,000 rpm the
gradient was punctured at the bottom and collected in 14 drop fractions.
Ten .mu.l from every third fraction was run on a 0.4% agarose gel and
compared to the lambda standards. Fractions 19 to 23 of the MboI
digestions were pooled into a 10-30 kb fraction and fractions 16 to 18
were pooled into >30 kb fraction. Fractions 23 to 26 of the Sau3A
digestions were pooled into the 10-30 kb fraction and fractions 15 to 22
were pooled into a >30 kb fraction. The pooled fractions were dialysed
against TE at 4.degree. C. for 48 hours, concentrated to 2 ml with
2-butanol, and precipitated with ammonium acetate and ethanol at
-20.degree. C. overnight. The DNA was resuspended in 400 .mu.l of TE, and
phenol extracted including a final ethyl ether extraction, ethanol
precipitated and resuspended in 100 .mu.l of TE.
(3) Preparation of pIJ922.
pIJ922 was cleaved at its unique BamHI site and treated with calf intestine
alkaline phosphatase (CIAP, Boehringer Mannheim, Indianapolis, Ind.).
pIJ922 DNA was isolated from S. lividans TK54 by the rapid boiling method,
as described in Example 1, from 4 liters of cells. Both TK54 and pIJ922
were obtained from D. Hopwood, John Innes Institute, Norwich England. 233
.mu.g of pIJ922 DNA were obtained. pIJ922 was cleaved by BamHI in a
reaction volume of 400 .mu.l containing 40 .mu.l of 10.times.KCl buffer,
(200 mM Tris, 200 mM KCl, 100 mM MgCl.sub.2, 1 mg/ml bovine serum albumin,
pH 7.9)100 .mu.l of pIJ922 (23.3 .mu.g), 5 .mu.l of BamHI restriction
enzyme (10 U/.mu.l), and 255 .mu.l of H.sub.2 O. After digestion at
37.degree. C. for 4 hours the DNA was treated with phenol and ethanol
precipitated. The DNA pellet was resuspended in CIAP reaction mixture
consisting of 100 .mu.l of 10.times.CIP Buffer (0.5M Tris pH 9.0, 10 mM
MgCl.sub.2, 1 mM ZnCl.sub.2, 10 mM spermidine) and 886 .mu.l of H.sub.2 O.
The reaction was begun by the addition of 7 .mu.l of CIAP, 28 U/.mu.l,
after incubation at 37.degree. C. for 30 minutes an additional 7 .mu.l of
CIAP was added. The reaction was terminated by the addition of 1 ml of
stop mix (10.times.STE 200 .mu.l [100 mM Tris pH 8.0, 1M NaCl, 10 mM
EDTA], 10% SDS 100 .mu.l, 0.5M EDTA pH 8.0 80 ml, and H.sub.2 O 620 .mu.l)
followed by heating at 65.degree. C. for 10 minutes. The DNA was phenol
extracted, concentrated to 1 ml with 2-butanol, ethanol precipitated and
resuspended in 250 .mu.l TE, ethanol precipitated again and resuspended in
100 .mu.l of TE. Aliquots of the treated pIJ922 were run on 0.7% agarose
gels and compared to known amounts of lambda DNA and pIJ922 cut with
BamHI. This indicated that 12 .mu.g had been recovered after the above
treatments.
(4) Ligation of pIJ922 to S. avermitilis genomic fragments.
Before ligation, the pIJ922 and the S. avermitilis Sau3A fragments >30 kb
were coprecipitated by mixing 5 .mu.l of CIAP treated pIJ922, 15 .mu.l of
Sau3A fragments >30 kb, 10 .mu.l of 3M sodium acetate, 80 .mu.l of TE and
275 .mu.l of ethanol. After overnight incubation at -20.degree. C., the
DNA pellet was resuspended in a mixture of 174 .mu.l of H.sub.2 O, 25
.mu.l of 10.times.ligase buffer (0.5M Tris pH 7.4, 100 mM MgCl.sub.2, 10
mM spermidine, and 1 mg/ml BSA), 25 .mu.l of 0.1M dithiothreitol, and 25
.mu.l of 10 mM ATP). The ligation was begun by the addition of 1 .mu.l of
T4 DNA ligase (New England Biolabs, Beverly Ma) and incubation was at
13.degree. C. overnight. In three experiments, S. lividans protoplasts
were transformed with 10 .mu.l of the ligation mix as described in Example
1, over 10,000 transformants were obtained. Among the transformants 4 were
observed to produce melanin, a pigment produced by S. avermitilis but not
S. lividans. The transformants were allowed to sporulate and the spores
were collected. When the spores were plated for single colonies on R2YE
and plasmid DNA was prepared it was observed that over 65% of the colonies
contained inserts of an average size of about 20 kb. The spores were used
to inoculate YEME medium and plasmid DNA was prepared by the rapid boiling
method and purified as described in Example 1. This pIJ922-S. avermitilis
library was used to isolate clones which complemented S. avermitilis
avermectin mutants.
B. Isolation of pAT1, a plasmid with the gene for C-5 avermectin
O-methyltransferase.
Protoplasts of MA6233, a strain deficient in C-5 avemectin
O-methyltransferase (OMT-) and which makes predominantly avermectin B1a
and B2a were transformed with the pIJ922-S. avermitilis library. The
transformation mixture was plated on RM14 regeneration medium and
incubated at 28.degree. C. After overnight incubation Thio.sup.r
transformants were selected by adding a 3 ml overlay of RM14 medium with
0.6% agar and containing 165 .mu.g/ml of thiostrepton. After 12 to 16 days
further incubation at 28.degree. C. the transformants were individually
patched onto sporulation Medium D using sterile toothpicks. After a
further 5-7 days of incubation at 28.degree. C., sporulation was evident.
Next, a 0.25 inch filter disk (Schleicher & Schuell, analytical paper
#740-E) was wetted with growth Medium E, rubbed across the sporulated
patch and used to inoculate production Medium F. After 12-16 days
incubation at 28.degree. C., the mycelia were extracted with methanol,
aliquots of the extract were applied to E. Merck Silica Gel 60 F-254 TLC
plates and the avermectins developed for 14 minutes with a
dichloromethane:ethyl acetate:methanol 9:9:1 solvent mixture. This solvent
system resolves the 8 avermectins into four spots; the avermectin a and b
components are not resolved and the order from fastest to slowest band is:
avermectin A1, A2, B1, and B2. Under these conditions MA6233 produces two
spots representing avermectin B1a+b and avermectin B2a+b. Over 10,000
transformants were screened for production of avermectin A1a+b and A2a+b.
An isolate which contained pAT1 was found to produce four spots which
co-chromatographed with avermectins A1a+b, A2a+b, B1a+b, and B2a+b.
Plasmid DNA was isolated from this isolate and used to transform MA6233
and 5 other S. avermitilis mutants defective in C-5 O-methyltransferase.
All six regained the ability to produce avermectin A1a+b and A2a+b. HPLC
analysis of methanol extracts from mutants containing pAT1 confirmed the
presence of avermectin A1a+b and A2a+b. In addition, the C-5
O-methyltransferase activity was measured in MA6233 with pAT1 and compared
to MA6233 containing the pIJ922 vector and an OMT.sup.+ S. avermitilis
strain containing pIJ922. MA6233 has less than 5% of the C-5
O-methyltransferase activity of the OMT.sup.+ strain, but MA6233
containing pAT1 had over 80% of the C-5 O-methyltransferase activity of
the OMT.sup.+ strain. This conclusively demonstrates that pAT1 contains
DNA which complemented the mutation in the OMT.sup.- strains tested and
presumably encodes the gene for C-5 O-methyltransferase.
______________________________________
Medium E
MgSO.sub.4.7H.sub.2 O (12.5% solution)
4 ml
NaCl (12.5% solution) 4 ml
MnSO.sub.4.H.sub.2 O (0.5% solution)
1 ml
ZnSO.sub.4.7H.sub.2 (1.0% solution)
1 ml
CaCl.sub.2.2H.sub.2 O (2.0% solution)
1 ml
FeSO.sub.4.7H.sub.2 O 25 mg
KNO.sub.3 2 g
Hy-Case SF (Humpko) 20 g
Yeast Extract (Difco) 20 g
Glucose 20 g
Tween 80 100 mg
Distilled water, add to a final
1000 ml
volume of
Adjust pH to 7.0 with NaOH
Medium F
Peptonized Milk 20 g
Ardamine pH 4 g
Glucose 90 g
MgSO.sub.4.7H.sub.2 O 0.5 g
CuSO.sub.4.5H.sub.2 O 0.06 mg
ZnSO.sub.4 6.sub.2 O 1 mg
CoCl.sub.2.6H.sub.2 O 0.1 mg
FeCl.sub.2.6H.sub.2 O 3 mg
Agar 15 g
Distilled water, add to a final
1000 ml
volume of
______________________________________
Adjust to pH 7.2 with NaOH. After autoclaving add 4 ml of filter sterilized
cyclohexamide solution (2.5 mg/ml) and 0.5 ml of thiostrepton solution (10
mg/ml in dimethyl formamide).
C. Characterization of pAT1.
pAT1 has a insert of about 20 kb, and a restriction map was determined for
pAT1 which is indicated in FIG. 1 and Table 3. pAT1 was introduced into 5
other OMT.sup.- mutants and all were then able to make substantial amounts
of avermectins with the O-methoxy at C-5. The location of the avrA gene
was determined to reside on the 3.4 kb BamHI fragment located between
11.13 kb and 14.53 kb of the restriction map of pAT1. This was determined
by subcloning the 3.4 kb fragment into the BamHI site of pIJ922 to
construct pAT83. When pAT83 was introduced into MA6233, it also allowed
the synthesis of avermectins A1a+b, A2a+b, B1a+b, and B2a+b.
TABLE 3
______________________________________
Restriction sistes in pAT1.
Site Site Interval Co-ordinate
# Name (bp) (bp)
______________________________________
1 EcoR I 1 1
2 BamH I 590 590
3 Bgl II 120 710
4 Xho I 810 1520
5 EcoR V 80 1600
6 Sph I 150 1750
7 Sst I 460 2210
8 Sca I 130 2340
9 Bgl II 350 2690
10 BamH I 400 3090
11 Sca I 470 3560
12 BamH I 230 3790
13 Xho I 1510 5300
14 Xho I 650 5950
15 BamH I 280 6230
16 BamH I 4900 11130
17 Pst I 1740 12870
18 Sph I 580 13450
19 Sst I 1030 14480
20 BamH I 50 14530
21 Pst I 100 14630
22 BamH I 2000 16630
23 BamH I 550 17180
24 Xho I 200 17380
25 Sst I 500 17880
26 Xho I 950 18830
27 Sst I 750 19580
28 Pst I 100 19680
29 EcoR I 300 19980
30 Bgl II 1000 20980
31 Xba I 4030 25010
32 Sph I 470 25480
33 Sst I 160 25640
34 Nde I 930 26570
35 EcoR V 505 27075
36 Bgl II 1375 28450
37 Pst I 910 29360
38 Sca I 980 30340
39 Sph I 210 30550
40 Sph I 370 30920
41 Sst I 1670 32590
42 Sca I 770 33360
43 Sph I 2380 35740
44 Sph I 6710 42450
45 Xho I 970 43420
46 EcoR I 630 44050
______________________________________
EXAMPLE 3
Isolation and characterization of pVE650
Plasmid pVE650 was isolated from the pIJ922-library. Protoplasts were
prepared from S. avermitilis mutant MA6278 (AGL.sup.-, OMT.sup.-). 200
.mu.l of protoplasts were transformed with 5.mu.l of TE containing about
25 ng of the library DNA. The transformation mixture was diluted and
plated on RM14 regeneration medium. After 20 hours incubation at
27.degree. C., the plates were overlayed with 3 ml of RM14 containing
165.mu.g/ml of thiostrepton and the incubation continued for 11 days. The
transformation plates were placed at 4.degree. C. and later, single
colonies were picked with a sterile toothpick on to sporulation Medium D.
After a further 5-7 days of incubation at 27.degree.-28.degree. C.,
sporulation was evident. Next, a 0.25 inch filter disk (Schleicher &
Schuell, analytical paper #740-E) was wetted with growth Medium E, rubbed
across the sporulated patch and used to inoculate production Medium F.
After 12-16 days incubation at 27.degree.-28.degree. C., the mycelia was
extracted with methanol, aliquots of the extract were applied to E. Merck
Silica Gel 60 F-254 TLC plates and the avermectins developed for 14
minutes with a dichloromethane:ethylacetate:methanol 9:9:1 solvent
mixture. Under these conditions MA6278 produces four spots representing
avermectin aglycones. The order, from fastest to slowest band is,
avermectin aglycone A1a+b, A2a+b, B1a+b, and B2a+b. (Although MA6278 is
OMT.sup.- it retains low C-5 O-methyltransferase activity and this
methylase apparently methylates the avermectin aglycones A1a+b and A2a+b
more efficiently than the glycosylated avermectin). Over 3000
transformants were screened for production of glycosylated avermectins. An
isolate which contained pVE650 was found to produce two spots which
co-chromatographed with avermectins B1a+b and B2a+b. Plasmid DNA was
isolated from this isolate and used to transform MA6278 and 25 other S.
avermitilis mutants defective in synthesizing or attaching oleandrose to
avermectin aglycone. Twenty-one regained the ability to produce
avermectins containing oleandrose.
A restriction map of pVE650 was determined and is indicated in Table 4 and
FIG. 2. The location of genes for synthesis or addition of oleandrose to
avermectin aglycones was determined by subcloning fragments from pVE650
into pIJ922 and introducing the resulting subclones into aglycone
producing mutants. Three complementation classes, representing at least
three genes, were discovered and are indicated in Table 2.
TABLE 4
______________________________________
Restriction sites in pVE650.
Site Site Interval Co-ordinate
# Name (bp) (bp)
______________________________________
1 EcoR I 1 1
2 BamH I 590 590
3 BamH I 2090 2680
4 BamH I 1820 4500
5 Sca I 1250 5750
6 Nru I 1000 6750
7 Stu I 1500 8250
8 BamH I 3250 11500
9 Pst I 1000 12500
10 BamH I 2000 14500
11 Bgl II 3250 17750
12 Pst I 350 18100
13 BamH I 1000 19100
14 EcoR I 570 19670
15 BamH I 810 20480
16 BamH I 20 20500
17 BamH I 530 21030
18 Bgl II 1000 22030
19 Bgl II 140 22170
20 BamH I 1080 23250
21 EcoR I 470 23720
22 BamH I 620 24340
23 Bgl II 390 24730
24 Xba I 4030 28760
25 Nde I 1560 30320
26 EcoR V 505 30825
27 Bgl II 1375 32200
28 Pst I 910 33110
29 Sca I 980 34090
30 Sca I 3020 37110
31 EcoR I 630 47800
______________________________________
EXAMPLE 4
A cloned avermectin gene alters the fermentation product composition: the
cloned avermectin O-methyltransferase gene.
The presence of the cloned avermectin O-methyltransferase (OMT) gene on a
plasmid in an avermectin producing strain alters the composition of the
avemectins produced. S. avermitilis strains containing the wild type
(unaltered) chromosomal OMT gene produce approximately 31% of the
avermectins as the A components with a methoxyl group at C-5 and
approximately 69% of the avermectins as the B components with a hydroxyl
group at C-5. The mutant strain, MA6233, deficient in avermectin
O-methyltransferase, produces only 4% of the avermectins as the A
components and 96% of the avermectins as the B components. When plasmid
pAT1, which contains the OMT gene, is transformed into the mutant strain
MA6233, the avermectin composition is restored almost to the wild type
strain levels with 26% of the avermectins as the A components and 74% of
the avermectins as the B components. When pAT1 is transformed into a
strain with a functional wild type OMT gene, the levels of the avermectin
A components is significantly increased to 66% while the proportion of the
avermectin B components is lowered to 34%. These experiments provide an
example of how the presence of a cloned gene in an avermectin producing
strain can alter the fermentation product composition resulting in an
efficient process to produce avermectin A1 and A2.
pAT1, when transformed into other Streptomyces strains that produce
secondary metabolites, can alter these fermentations in a similar manner
to the first example and result in the production of methylated
derivatives of the natural fermentation product. These new and novel
derivatives may be more potent and have improved activity spectra.
EXAMPLE 5
Isolation of other genes for avermectin biosynthesis
Ikeda et al have demonstrated that the genes for the synthesis of the
avermectin aglycone are genetically linked to the genes for synthesis or
attachment of the oleandrose moiety to avermectin aglycone. Thus, the
other genes for avermectin biosynthesis can be cloned by isolating DNA
adjacent to the insert DNA of pAT1 and pVE650 clones.
DNA adjacent to the insert in pVE650 was isolated from a cosmid library of
S. avermitilis DNA. The cosmid vector used was pVE328, a cosmid vector
which can replicate in E. coli conferring ampicilin-resistance (Amp.sup.r)
and Streptomyces conferring Thio.sup.r. pVE328 is only 7.5 kb in size so
it can clone DNA fragments up to 43 kb, and pVE328 contains two lambda cos
sites so it can be efficiently packaged into phage heads in vitro. pVE328
also contains unique Bg1II and HpaI cloning sites flanked by DraI sites.
The Bg1II cloning site allows the incorporation of fragments with GATC
ends, which are produced by BamHI, Bg1II, Bc1I, XhoII, and MboI. The HpaI
site can be used to clone blunt ended fragments. Since Streptomyces DNA
has a high G+C ratio, often greater than 70%, the DraI site TTTAAA is very
rare (1 per 300 kb). Thus, most fragments cloned into the Bg1II or HpaI
sites can be excised with DraI for further analysis and manipulation.
Finally the vector contains the broad host range Streptomyces phage TG1
cos site. This site can be used in vivo by TG1 helper phage to package
pVE328 derivatives into TG1 virions. A TG1 lysate grown on a pVE328
derivative can then be used to introduce the pVE328 derivatives into other
Streptomyces by phage mediated transduction. This technique is technically
simpler than transformation and expands the hosts into which the clone may
be introduced.
pVE328 was constructed using standard recombinant DNA technology differing
little from the procedures in Maniatis et al., supra.
The starting plasmid was pSVO10X2 obtained from Rick Myers via F. Foor.
pSVO10X2 is a deletion derivative of pBR322 which contains two multiple
cloning regions and some SV40 DNA. A derivative of pSVO10X2 which
contained a single multiple cloning region and lacked the SV40 DNA was
isolated after a complex ligation. This ligation involved two digestions
of pSVO10X2 (one with PstI, HindIII and PvuI and the other with BamHI,
HindIII and PvuI), and the digestion of pMC1403 (obtained from M.
Casadaban, University of Chicago), with BamHI, PstI, and PvuII. Among the
products of this ligation was a 2 kb plasmid, designated pVE61, that was
found to have a multiple cloning region containing sites for the enzymes
EcoRI, SmaI, BamHI, PstI, Bg1II, XbaI, and HindIII. This plasmid was
converted to an E. coli lambda cosmid by addition of 405 bp HincII cos
fragment from pVE81, into the unique SmaI site of pVE61 to yield pVE105.
The cos region in pVE 81 had been previously cloned as a 3.2 kb
EcoRI-Bg1II fragment from lambda into the EcoRI-BamHI sites of pBR322 to
yield pVE81. The PstI site in the amp gene of pVE105 was removed by
substituting the Bg1I to AatII fragment of pUC8 (Bethesda Research
Laboratories, Gaithersburg, Md.) for the Bg1I to AatII fragment of pVE105
to yield pVE163. This cosmid was made into a shuttle cosmid by ligation of
the Streptomyces plasmid pVE95 to pVE167. pVE95 is a stable, Thio.sup.r
deletion derivative of pVE28 isolated after digestion of pVE28 with SstI
and subsequent ligation. pVE95 was linearized at its unique Bg1II site and
ligated to pVE163 linearized at its unique BamHI site to yield pVE167.
Unique cloning sites were introduced into pVE167, linearized at its Bg1II
site by the ligation of a synthetic oligonucleotide of sequence:
##STR1##
The double strand oligonucleotide was prepared by mixing together 10 .mu.g
of each single stranded 29 mer in 50 .mu.l of TE, the mixture was heated
to 85.degree. C. for 5 minutes, slowly cooled to room temperature and
stored overnight at 4.degree. C. One half microgram of pVE167 was mixed
with a 50 fold molar excess of annealed oligonucleotide and ligated with
T4 DNA ligase. Among the Amp.sup.r transformants, an isolate containing a
single copy of the oligonucleotide was identified and designated pVE232.
pVE232 was converted into a Streptomyces phage TG1 cosmid by addition of a
270 bp EcoRV-HpaI fragment containing the TG1 cos site. pVE232 was
linearized at its XbaI site, the site made blunt by treatment with DNA
polymerase Klenow fragment (Bethesda Research Laboratories, Gaithersburg,
Md.), and ligated to TG1 cleaved with HpaI and EcoRV. pVE288 was
identified as derivative which contained the 270 bp cos fragment. The TG1
cos fragment can be cleaved from pVE288 with XbaI since insertion of the
HpaI-EcoRV fragment into the filled in XbaI site regenerated two XbaI
sites. A second lambda cos site was inserted into a pVE288 after it was
digested with EcoRI and treated with DNA polymerase Klenow fragment. The
resulting blunt-ended linear molecule of pVE288 was ligated to pVE81
digested with HincII and a derivative with two lambda cos sites in the
same orientation was identified and designated pVE328. The primary cloning
site, Bg1II, is indicated by ** in FIG. 4.
A library of S. avermitilis DNA was prepared in the cosmid vector pVE328.
Partially digested Sau3A treated S. avermitilis chromosomal DNA was
separated on a 15-40% sucrose gradient. Fractions containing fragments
from 35 to 45 kb were pooled, the sucrose was removed by dialysis against
TE buffer, and the fragments were concentrated by ethanol precipitation.
About three micrograms of fragments were mixed with 0.5 or 0.2 .mu.g of
pVE328 in a total volume of 20 .mu.l. The pVE328 DNA had been previously
cleaved at its single Bg1II site and treated with CIAP. After ligation at
12.degree. C. for 16 hours with T4 DNA ligase, 4 .mu.l of the DNA mixture
was packaged into phages with an Ambersham lambda in vitro packaging kit.
The cosmid library was transduced into E. coli strain RR1 selecting
Amp.sup.r. 2016 transductants were picked individually into cells of
microtiter dishes containing 0.15 ml of LB medium with 75 .mu.g/ml of
ampicillin, grown overnight at 37.degree. C., 15 .mu.l of dimethyl
sulfoxide was added, the plates were sealed in Seal-A-Meal bags and quick
frozen in a dry ice/ethanol bath and stored at -80.degree. C. This was the
cosmid library of S. avermitilis.
Filters containing DNA from the cosmid library were prepared by growing a
replica of the library on LBamp agar (75 .mu.g/ml of ampicillin). Before
cultures of the 2016 clones were frozen, 5 .mu.l aliquots from each
culture were transferred to LB amp agar (75 .mu.g/ml of ampicillin) in an
8 by 12 pattern. After overnight incubation at 37.degree. C., an ICN
Biotrans nylon membrane (1.2 micron rating) was placed on the colonies for
1 hour and incubation continued at 37.degree. C., then the filters were
transferred, colony side up, to LB-amp agar for 4 hours of further
incubation at 37.degree. C. The filters were then transferred to
LB-amp-cam agar (50 .mu.g/ml chloramphenicol) and incubated overnight at
37.degree. C. DNA was released from the cells and fixed to the filters by
transferring the filters to a series of Whatmann 3 MM filters saturated
with various solutions. Between each transfer the filters were placed on
dry 3 MM paper to blot off excess solutions. The filters were transferred
to 3 MM saturated with 10% SDS for 5 minutes at room temperature, then the
cells were lysed by transferring the filters to 3 MM saturated with 0.5 N
NaOH, 1.5 N NaCl for 5 minutes at room temperature and then placed in a
100.degree. C. steam cabinet for an additional 5 minutes. The filters were
neutralized by transfer to 3 MM saturated with 0.5M Tris, pH 7.9, 1.5N
NaCl and incubated at room temperature for 5 minutes. The filters were
then immersed in a solution of 2.times.SSC (SSC is 0.15M NaCl, 0.015M
trisodium citrate, pH 7.0) for 2 seconds, then immersed in 95% ethanol for
10 seconds, air dried, and baked at 80.degree. C. for 1 hour in a vacuum
oven. The residual cell debris was removed by three washings of the
filters at 65.degree. C. in 0.1% SDS, 3.times.SSC, each wash for 15
minutes. After washing, the filters were dipped in 2.times.SSC, air dryed
on 3 MM paper and saved at 4.degree. C. until used.
Twenty-one filters each containing DNA from 96 cosmid clones was probed
using the 1.09 kb BamHI fragment from one end of plasmid pVE650. This DNA
was labelled with 32-P dCTP using a random priming kit (U.S. Biochemicals,
Cleavland, Ohio). The purified 1.09 kb BamHI fragment (0.5 .mu.g) in 5
.mu.l of TE was denatured by heating at 95.degree. C. for 10 minutes and
then chilling on ice for 10 minutes. The following were added to the DNA:
3 .mu.l of a 1:1:1 mixture of dATP:dTTP:dGTP, 2 .mu.l of 10.times.reaction
mixture containing the random hexanucleotides, 3 .mu.l of H.sub.2 O, 5
.mu.l of [.sup.32 P] dCTP (specific activity of 3000 Ci/mmole), and 1
.mu.l of klenow enzyme. The mixture was mixed, microfuged for 10 seconds,
then incubated for 30 minutes at 37.degree. C. The reaction was terminated
by adding 2 .mu.l of 0.25M EDTA, pH 8. The labelled 1.09 kb fragment was
passed through a Centri-Sep Column (Princeton Seperations, Adelphia,
N.J.)by centrifugation at 12,000 rpm for 3 minutes at 4.degree. C. Each of
the 21 filters was put into a separate plastic Seal-A-Meal bag #6006
(Dazey Corp., Industrial Airport, Kans.), 9.5 ml of prehybridization
solution was added, and the bag heat sealed. Prehybridization solution
contained 0.75M NaCl, 0.075m NaCitrate, pH 7.0, 0.1% Ficoll, 0.1%
polyvinylpyrrolidone, 0.1% BSA, 50% formamide, 0.1% SDS, and 100 .mu.g/ml
of sheared herring sperm DNA that was heat denatured for 10 minutes in a
boiling water bath. After a 3 hour incubation at 43.degree. C., the
prehybridization solution was removed and 12 ml of a hybridization
solution was added to the same bag containing each of the 21 filters. The
hybridization solution was modified prehybridization solution that
contained 10% dextran sulfate.
The probe was heated at 95.degree. C. for 10 minutes then put on ice. A
total of 2.times.10.sup.6 counts was added to each hybridization bag and
the bags were heat sealed. After sealing an effort was made to distribute
the probe evenly throughout the hybridization solution. The hybridization
was carried out at 43.degree. C. overnight in a water bath with slow
agitation. After 18 hours the filters were removed from the bags and
rinsed two times in 0.3M NaCl, 0.03M NaCitrate, pH 7.0, 1% SDS at room
temperature. The filters were then washed twice in 0.3M NaCl, 0.03M sodium
citrate, pH 7.0, 0.1% SDS for 15 minutes at 43.degree. C. The filters were
then washed twice in 0.015M NaCl, 0.0015M sodium citrate, pH 7.0, 0.1% SDS
for 10 minutes at 43.degree. C., and twice in 0.015M NaCl, 0.0015M sodium
citrate, pH 7.0, 0.1% SDS for 10 minutes at 60.degree. C. All the filters
were blotted on Whatman 3 MM paper and exposed to X-ray film (Kodak X-OMAT
AR-5) for 14 days. This initial screen yielded 81 putative clones.
The individual cosmid cultures that yielded a positive signal to the 1.09
kb BamHI fragment were spotted in triplicate on LB plates containing 100
.mu.g/ml of ampicillin and incubated overnight at 37.degree. C.,
refrigerated for 2 hours at 4.degree. C., a 82 mm nitrocellulose filter
(Schleicher and Schuell, BA85, 0.45 micron) placed on the plates for 2
minutes, and then the filter containing bacteria was placed on LB agar
plates containing 10 .mu.g/ml of chloramphenicol with the colony side up.
The plates were incubated for 12 hours at 37.degree. C. and then the
bacteria were lysed and the DNA fixed to the filters. The bacterial
colonies on the filters were lysed by laying the filters, colony side up,
on a sheet of Whatman 3 MM paper soaked with 0.5M NaOH for 3 minutes. Next
the filters were moved to another sheet of Whatman 3 MM paper containing
0.5M NaOH and left for an additional 3 minutes. The filters were then
transferred to Whatman 3 MM paper containing neutralization buffer (1.0M
TrisHCl pH8/1.5M NaCl) for 3 minutes. The above step was repeated. The
filters were removed and placed on Whatman 3 MM paper and allowed to air
dry for 30 minutes. The dried filters were sandwiched between two sheets
of 3 MM paper and baked for 45 minutes at 80.degree. C. in a vacuum oven.
The baked filters were then hybridized with 32-P labelled 1.09 kb BamHI
fragment as described above. Of the 81 initial putative clones, 9 colonies
gave a positive signal on all three filters. The nine cosmid clones were
grown in liquid culture to isolate large amounts of purified DNA for
restriction analysis.
The 21 filters containing the cosmid libary described above were stripped
of the hybridized probe and probed with the 2.09 kb BamH I fragment from
the other end of pVE650. The filters, which were not allowed to dry, were
stripped of the probe by washing 2 times for 20 minutes in 500 ml of
0.015M NaCl, 0.0015M NaCitrate, pH 7.0, 0.5% SDS at 95.degree. C. The
filters were exposed to X-Ray film for 48 hours to insure that the probe
was removed. The 21 filters were then probed as described above for the
1.09 kb fragment except the probe was the 2.09 kb BamHI fragment. The
initial screen of the library yielded 93 putative cosmids that hybridized
with the 2.09 kb BamHI fragment from pVE650. Upon retest, 12 of the
cosmids were positive and DNA was purified from the 12 clones.
The various cosmid clones were mapped by restriction analysis relative to
the BamHI fragments in pAT1 and pVE650. In addition a Southern analysis
was performed to identify which clones contained fragments from the Group
1 and Group 2 homology groups. This allowed the identification of 4 clones
which collectively represent over 110 kb of genomic DNA. Their location
relative to pAT1 and pVE650 is indicated in FIG. 3. To test if these
clones contain DNA for other avermectin genes, fragments were subcloned
from the cosmids onto pIJ922. One subclone, pVE941, contained a 14 kb
Bg1II fragment from pVE859. This DNA was transformed into aglycone
producing mutants that were not complemented by pVE650. All five mutants
regained the ability to produce glycosylated avermectins. In addition,
this DNA was introduced into MA6316 (GMT.sup.-), and MA6323 (GMT.sup.-
OMT.sup.-) mutants which do not methylate the 3' and 3" hydroxyls of
avermectin. These mutants were also complemented (Class GMT in Table 2).
The genes for glycosylation on pVE650 and pVE941 can be subcloned onto a
single plasmid. A restriction digestion of pVE859 with PstI produced 8
bands. The largest PstI fragment was gel purified and cloned into pVE1043
to form pVE1116. (pVE1043 was derived from pIJ922 in two steps. First,
pVE1023 was made by destroying the PstI site in pIJ922 by cleaving pIJ922
with PstI and filling in that site using T4 DNA polymerase. pVE1043 was
constructed by inserting a synthetic oligonucleotide into pVE1023 digested
with EcoRI and BamHI. The oligonucleotide consisted of the sequence:
##STR2##
This resulted in the formation of a polycloning site with unique sites for
EcoRI, HpaI, PstI, NheI, AseI, HindIII, DraI, and BamHI.) pVE1116
complemented all Class III, Class IV, Class V, Class VI and GMT mutants.
In addition it was confirmed to complement a representative mutant from
Class I and Class II. Thus, it appears that pVE1116 contains all the genes
for glycoyslation of avermection. This plasmid will allow the
biotransformation of avermectin aglycones into avermectin. When this
plasmid is introduced into other strains producing antibiotics which
contain an appropriate free hydroxyl, this plasmid will add oleandrose to
the antibiotics to make novel antibiotics. These novel antibiotics may
have enhanced activity.
A comparison of the restriction maps of pAT1, pVE923 and pVE924 showed that
the region adjacent to the 0.55 kb BamHI fragment was different in the
three clones. On pAT1, a 3.4 kb BamHI to vector junction fragment, which
contains an EcoRI site, maps adjacent to the 0.55 kb BamHI fragment. On
pVE924 a 3.2 kb BamHI fragment without an EcoRI site is located adjacent
to the 0.55 kb BamHI fragment. Cosmid pVE923 has a 7.0 kb BamHI fragment
located adjacent to the 0.55 kb BamHI fragment. In order to determine the
actual structure of this region of the avermectin gene cluster, DNA form
the S. avermitilis chromosome was directly cloned into E. coli.
The method chosen to directly clone DNA from S. avermitilis into E. coli
relies on the homologous recombination system of S. avermitilis to direct
the integration of an E. coli plasmid. The E. coli plasmid contains two
fragments of the avemectin cluster which flank a region of interest. Such
a plasmid will integrate by recombination between the genome and one of
the homologous fragments. The resulting integrant has a duplication of
each region represented by the two cloned fragments. Recombination between
the duplicated regions will result in excision of the vector. If this
recombination occurs between a different region than the recombination
which resulted in integration, then the resulting excision plasmid will
contain the two cloned fragments and all the DNA between them.
Any E. coli vector which does not replicate in Streptomyces can be used if
it has the following features: a gene foe selection in E. coli, a gene for
selection in Streptomyces, and unique sites for cloning two fragments. For
these experiments a derivative of pVE616 was made. pVE616 already
contained a gene for selection E. coli (amp) and a gene for selection in
Steptomyces (thio). A synthetic oligonucleotide was made to provide useful
cloning sites of the following sequence:
##STR3##
The oligonucleotide was cloned into the BamHI to PstI sites of pVE616
resulting in a pVE1011 with a polycloning site for BamHI, HpaI, Bg1II,
SstI and PstI. FIG. 7 displays a restriction map of pVE1011. The two
fragments chosen for cloning into pVE1011 flank the 0.55 kb BamHI
fragment. The 3.4 kb BamHI fragment of pAT1 and the 3.7 kb BamHI fragment
from pVE924 were chosen (see Table 6). The 3.4 kb BamHI fragment of pAT1
was purified from an agarose gel and ligated to Bg1II digested, CIAP
treated pVE1011. After transformation of E. coli, Amp.sup.r transformants
were screened for the insert. One transformant, contained a plasmid with a
3.4 kb insert and the plasmid was designated pVE1038. pVE1038 was isolated
from 500 ml of LBamp (LB containing 100 .mu.g/ml of ampicillin) grown
culture and purified by CsCl banding. Next pVE1038 was digested with BamHI
and HpaI and ligated to a gel purified, 2.9 kb BamHI-HpaI fragment of Tn5.
The resulting Neo.sup.r Amp.sup.r transformant contained a plasmid
pVE1051, with the 2.9 kb fragment. pVE1051 was isolated from 500 ml of
LBamp grown culture and purified by CsCl banding. Next pVE1051 was
digested with BamHI, treated with CIAP, and ligated to the gel purified
3.7 kb BamHI fragment of pVE924. A transformant was identified with the
3.7 kb insert and the orientation of the 3.4 kb and 3.7 kb BamHI fragments
were the same as in the chromosome. The DNA of the plasmid, designated
pVE1299, was transformed into the DNA methylation deficient strain,
MB5386. S. avermitilis has a methyl specific restriction system (J. Bact.
170 pg 5607-5612 (1988)). Thus, before DNA can be introduced into S.
avermitilis from E. coli it must be isolated from a strain deficient in
dam and dcm methylation. Five .mu.g of CsCl purified DNA of pVE1299,
isolated from MB5386, was introduced into 100 .mu.l of S. avermitilis
protoplasts. Transformants were selected as Neo.sup.r Thio.sup.r and one,
designated GG1776, was saved. Small scale plasmid preparations were made
from 6 ml of GG1776 grown in YEME with 5 .mu.g/ml thiostrepton and with 5
.mu.g/ml neomycin. Ten microliters of the resulting DNA preparation was
used to transform E. coli and Amp.sup.r Neo.sup.s transformants were
examined. As expected, these transformants contained the 3.4 kb, 2.1 kb,
0.55 kb BamHI fragments, as well as the 7.0 kb BamHI band of pVE923.
Surprisingly the transformants also contained two new BamHI fragments of
8.0 kb and 7.4 kb. The 7.0 kb, 8.0 kb, and 7.4 kb fragments are absent
from pVE924 and pAT1. Thus pVE924 contained a deletion of DNA between the
0.55 kb and 3.7 kb BamHI bands resulting in the 3.2 kb BamHI fragment. One
transformant, ET14167, with a plasmid designated pVE1446, was saved.
Restriction mapping then established the order of fragments on pVE1446
(see Table 6). It is likely the DNA represented by the 7.4 kb and 8.0 kb
BamHI fragments contains avermectin genes since avermectin genes have been
located on either side of this region. The E. coli strain containing
pVE1446 has been designated MB5472 and deposited as ATCC 68250.
TABLE 5
__________________________________________________________________________
Size of BamHI fragments on plasmids
containing S. avermitilis DNA in kilobase pairs.
PVE923
PAT1 pVE1446
pVE924
pVE855
pVE650
pVE859
__________________________________________________________________________
17.50 27.45
8.00 9.10 10.00
24.05
13.60
7.40 4.90 7.60 5.50 7.40 7.40 5.70
7.00 3.40 7.40 4.90 5.60 4.70 5.50
3.60 2.50 7.00 3.70 4.70 3.00 4.70
2.10 2.44 3.70 3.40 3.30 2.35 4.00
1.75 2.10 2.10 3.15 3.00 2.09 3.20
1.50 0.70 0.55 2.60 2.60 1.85 3.00
0.85 0.55 2.44 2.50 1.40 2.35
0.58 2.30 2.35 1.09 1.65
0.55 2.10 1.85 0.53 1.40
1.90 1.60 0.02 0.53
1.30 1.40 0.02
1.20 0.95
0.95 0.75
0.75 0.53
0.55 0.02
__________________________________________________________________________
TABLE 6
______________________________________
Restriction map of the avermectin gene cluster.
Site Site.sup.1 Interval Co-ordinate
# Name (bp) (bp)
______________________________________
1 BamHI 1300 1300
2 BamHI 2300 3600
3 PstI 1740 5340
4 BamHI 1660 7000
5 PstI 100 7100
6 BamHI 2000 9100
7 BamHI 550 9650
8 EcoRI 6800 16450
9 BamHI 200 16650
10 StuI 4300 20950
11 BamHI 3100 24050
12 StuI 7700 31750
13 BamHI 300 32050
14 BamHI 3700 35750
15 BamHI 4900 40650
16 BamHI 1200 41850
17 BamHI 2440 44290
18 BamHI 750 45040
19 BamHI 950 45990
20 BamHI 5500 51490
21 BamHI 2600 54090
22 BamHI 1900 55990
23 ScaI 100 57690
24 StuI 2300 59990
25 ScaI 1300 61290
26 BamHI 1700 62990
27 PstI 2000 64990
28 BamHI 1000 65990
29 BglII 3350 69340
30 PstI 350 69690
31 BamHI 1000 70690
32 EcoRI 570 71260
33 BamHI 830 72090
34 BamHI 20 72110
35 BamHI 530 72640
36 BglII 1130 73770
37 BglII 140 73910
38 StuI 980 74890
39 BamHI 100 74990
40 EcoRI 470 75460
41 BamHI 1180 76640
42 BamHI 200 76840
43 EcoRI 400 77240
44 BamHI 2800 80040
45 PstI 1290 81330
46 BamHI 2710 84040
47 BglII 3290 87330
48 BamHI 2410 89740
49 BamHI 5500 95240
______________________________________
.sup.1 The BamHI sites in this 95 kb region have been mapped. Only some o
the Bg1II, EcoRI, PstI, StuI, and ScaI sites have meen mapped.
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